Hyperoxia-induced changes in morphometric parameters of postnatal neurogenic sites in rat

The ability of stem cells to differentiate in to a variety of cell types is the fundamental basis for life in all organisms. But also later in life, the contribution and ability of stem cells to differentiate in tissue specific cells is crucial and needs to be preserved in adulthood. Yet, stem cells can only be found in a suitable environment called stem cell niches. The niche maintains the stem cell properties of the cells or provides factors required for their differentiation. In the adult brain, there are two distinct niches of adult neural stem cells (NSCs) that can be found in the subventricular zone (SVZ) on the outside walls of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus (DG). In the latter, NSCs differentiate into granular neurons and astrocytes that may contribute to learning and memory formation. This process is not only strictly regulated on a genetic level, but also post-transcriptional regulation of RNA transcripts strongly contributes to the extremely fine-tuned regulation of adult neurogenesis. A key factor in this process is the microprocessor, a complex best-known for its involvement in the microRNA (miRNA) pathway. The microprocessor consists of Drosha and DGCR8, and processes the pri-miRNA into pre-miRNA by targeting and cleaving distinct hairpin (HP) structures in these transcripts. The main effector of this process is the RNAse III Drosha. However, Drosha has been shown to participate in many additional processes that are not related to miRNA biogenesis and thus are referred to as non-canonical functions. Of special interest for this study is the finding that Drosha is able to target evolutionary conserved HPs that are located in the untranslated regions (UTRs) of mRNAs coding for transcription factors that drive NSCs into specific cell fates. In the adult hippocampal DG, Drosha targets and cleaves the 3’ UTR HP of the mRNA of NFIB, which leads to de destabilization and degradation of the whole mRNA transcript. Drosha thus prevents NFIB protein expression and hinders the cell to undergo oligodendrogenesis. This downregulation of NFIB is required to maintain the DG NSC pool and guarantee proper neuronal development and differentiation. Interestingly, NFIB contains two RNA HPs, located in the 5’ and 3’ UTRs. Both HPs are targeted by Drosha but only the 3’ UTR HP is cleaved. As Drosha is ubiquitously expressed in the entire brain, it remained to be shown how transcripts can escape Drosha cleavage and express NFIB in a cell-specific manner when needed, for example during oligodendrogenesis in other brain regions. In order to answer this question, we developed strategies to identify regulatory proteins that control Drosha-mediated NFIB cleavage in DG NSCs. By using protein immunoprecipitation (IP) followed by dual mass spectrometry (MS2), we identified Drosha interacting proteins in DG NSCs. Moreover, we also determined NFIB HP interacting proteins by RNA pulldown assays and MS2. This data did not only allow us to investigate closer some putative regulators of Drosha cleavage, but also allowed us to characterize the Drosha interactome in NSCs. Additionally, we analyzed the differences between NFIB 3’ UTR and 5’ UTR interaction partners and could show that especially the 5’ UTR HPs interacts closely with ribosomal protein and thus seems to be deeper involved in translation than the 3’ UTR HP. In order to investigate the functional relevance of the Drosha and NFIB mRNA bound proteins, we developed an in vitro GFP reporter assay in DG NSCs to directly monitor Drosha activity upon overexpression of putative cleavage regulating partners. We found that overexpression of certain candidate proteins modulates Drosha cleavage, especially gain-of-function of Scaffold Attachment Factor B1 (Safb1) significantly reduced the read-out GFP signal on mRNA as well as protein levels. This reduction was found to be Drosha-dependent as Drosha cKO in DG NSCs could rescue the reduced GFP levels caused by Safb1 overexpression. Moreover, Safb1 overexpression also reduced the mRNA level of endogenous NFIB. We could confirm direct binding of Safb1 to Drosha by protein IP and to NFIB by crosslinking and immunoprecipitation (CLIP) experiments. To investigate whether Safb1 gain-of-function and the consequent increase in Drosha cleavage has an influence of cell fate determination we overexpressed Safb1 in SVZ NSCs, an oligodendrogenic population, and found reduced oligodendrogenesis in the transfected cells. Thus we identified Safb1 as a key regulator of Drosha-mediated cleavage of NFIB mRNAs, and could show that Safb1 levels influence NSC differentiation and oligodendrogenesis.

It had long been held as dogma that no new neurons are added to the central nervous system after birth. The fixed neuronal population of the adult brain was understood to be necessary to maintain the functional stability of the adult brain and was also taken as an explanation for the lack of endogenous repair within the central nervous system following injury. However, there were early reports of cell proliferation in the adult brain (1–4), and these observations have been validated by the use of new, more powerful labeling techniques over the last decade (5). Nevertheless, under normal conditions, neurogenesis in the adult brain appears to be restricted to the subventricular zone and the hippocampal dentate gyrus. This review summarizes our current understanding regarding neurogenesis in the adult dentate gyrus, the extent to which primitive neural progenitor (stem) cells in the hippocampus can be manipulated in vitro and in vivo, and the implications of hippocampal neurogenesis for brain function and plasticity. Because of the importance of quantitative studies to our understanding of neurogenesis, some methodological and conceptual issues regarding histological quantitation are also addressed.

Objective To explore the effects of arsenic exposure on learning and memory and its potential mechanism in rats. Methods Water-based arsenic-exposed rat models were established on 4-10 postnatal days.The experimental animals were divided into 4 groups (10-12 cases in each group): the control group, the 15 μg/L As2O3 water group, the 30 μg/L As2O3 water group, and the 45 μg/L As2O3 water group.Cognitive functions were examined with the Morris water maze, exploratory behavior was detected by the exploratory behavior test.The hippocampus of pups from each experimental group was sectioned at various time points after arsenic exposure.The morphologies and neurogenesis of the neurons in the hippocampus CA1-CA3 region and dentate gyrus (DG) were observed by hematoxylin-eosin staining, Nissl staining, and doublecortin (DCX) immunostaining at different time points after arsenic exposure. Results Compared with the normal control group, the escape latency of the rats in the arsenic-exposed group was prolonged.The average escape latency of the rats in the normal control group, 15 μg/L As2O3 group, 30 μg/L As2O3 group and 45 μg/L As2O3 group were (17.00±9.53) s, (35.89±19.81) s, (26.60±18.84) s, and (33.79±18.08) s, respectively, and the difference among 4 groups was statistically significant (F=3.591, P<0.05), and the residence time in the original target quadrant was shortened, respectively, (38.93±8.33) s, (36.03±16.25) s, (29.85±9.27) s, and (29.84±10.16) s, respectively, and there was no significant difference among 4 groups (F=1.681, P=0.187). HE staining and Nissl staining showed that pathological changes such as edema, degeneration and necrosis were observed in the hippocampal CA1 area and CA2 area as well as dentate gyrus cells in rats exposed to arsenic in the acute phase.The higher the concentration of arsenic exposure, the more obvious the cell structure disorder was.However, 5 weeks after exposure, the pathological changes in hippocampal neurons in the arsenic-exposed group gradually returned to normal.Immunohistochemistry showed that the expressions of DCX in the CA1, CA2 and dentate gyrus of rats exposed to arsenic decreased significantly 24 h after arsenic exposure, especially in the 45 μg/L group.Five weeks after arsenic exposure, there was no expression in the hippocampal CA1-CA3 area, and there was still a small amount of expression in the dentate gyrus. Conclusions Postnatal low-concentration arsenic exposure may impair learning and abnormal germination of neurons in the hippocampal dentate gyrus may be the underlying mechanism. Key words: Arsenic exposure; Learning and memory; Hippocampus; Mechanism

Mesenchymal stromal cells (MSC) have been suggested to have beneficial effects on animal models of traumatic brain injury (TBI), owing to their neurotrophic and immunomodulatory properties. Adipose tissue-derived stromal cells (ASCs) are multipotent MSC that can be harvested with minimally invasive methods, show a high proliferative capacity, low immunogenicity if allogeneic, and can be used in autologous or heterologous settings. In the present study ASCs were genetically labelled using the Sleeping Beauty transposon to express the fluorescent protein Venus. Venus+ASCs were transplanted intra-cerebroventricularly (ICV), on a rat TBI model and their survival, fate and effects on host brain responses were examined at seven days post-injury (7dPI). We provide evidence that Venus+ASCs survived, migrated into the periventricular striatum and were negative for neuronal or glial lineage differentiation markers. Venus+ASCs stimulated the proliferation of endogenous neural stem cells (NSCs) in the brain neurogenic niches, the subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). It was also evident that Venus+ASCs modify the host brain's cellular microenvironment both at the injury site and at their localization area by promoting a significant reduction of the lesion area, as well as altering the post-injury, pro-inflammatory profile of microglial and astrocytic cell populations. Our data support the view that ICV transplantation of ASCs induces alterations in the host brain's cellular response to injury that may be correlated to a reversal from a detrimental to a beneficial state which is permissive for regeneration and repair.

Adult neurogenesis continues throughout life in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) of mammals. At the base of adult neurogenesis lie adult neural stem cells (NSCs). These cells can either be found in a dormant, non-dividing state (quiescent) or in a proliferating state (active). Over the last three decades the field of neurogenesis has expanded, but there are still open questions with regards to adult NSC maintenance and potential capacity. Over the course of my PhD studies I addressed three major questions of adult NSC maintenance. (1) What are the differences between active and quiescent NSCs? (2) Do NSCs have similar maintenance factors in the SVZ and the SGZ? (3) What are the capacities of distinct subtypes of NSCs and progenitors to respond to external stimuli? I was able to show that in the adult mouse brain, Notch2 is the gatekeeper of quiescent NSCs in both neurogenic niches, the SVZ and the SGZ. The loss of this Notch paralogue led to the activation of quiescent NSCS and a prolonged and abnormal activation, followed by NSCs exhaustion in the long term. If Notch1 was deleted in addition to Notch2, quiescent and active NSCs are no longer maintained properly and will differentiate to a neural fate. Thus an intricate interplay between Notch1 and Notch2 is needed for adult NSC maintenance in both neurogenic niches. In the SVZ the receptors Notch1 and Notch2 are coexpressed on NSCs. We addressed NSC identity also in the second neurogenic niche, the SGZ, where the receptors are also coexpressed by NSCs. The loss of Notch2 led to the activation of quiescent NSCs and an increased production of neuroblasts. The differential signal requirement for the maintenance of quiescent and active NSCs raises the question, whether these distinct cell populations might have unique functions in response to external physiological and/or pathological stimuli. In order to address this question we characterized the SGZ in great detail at different ages. In the geriatric SGZ active NSCs were lost and the NSCs that remained were quiescent. These quiescent NSCs have the capacity to replenish the active NSC pool upon induction of epileptic seizures. On the other hand, administration of antidepressants left the NSCs unaffected initially. It was the amplifying progenitor pool that responded. In long-chase experiments the NSCs were then reactivated by either the resulting induced changes from the amplifying progenitors or a delay in NSC response. NSC maintenance in the adult murine brain is an intricate mechanism highly dependent on the proper internal and external mechanisms. In the work presented here, I will illustrate the importance of Notch signaling in NSC maintenance and the high level of heterogeneity within the NSC pool and the NSC niche.

Evidence for the generation of young neurons out of precursor cells in the adult brain, i.e. adult neurogenesis, exists for at least two brain regions. New nerve cells are generated in the subventricular zone of the olfactory bulb and in the subgranular zone of hippocampal dentate gyrus. Young neurons of the subgranular zone migrate along the rostral migratory stream to the olfactory bulb, where they functionally integrate and contribute to the discrimination of odors. In the hippocampus the function of newly formed granule cells is still a matter of debate, yet it is thought that adult neurogenesis functionally contributes to hippocampal functions. Over the last twenty years of extensive research it became clear that adult hippocampal neurogenesis (AHN) in laboratory rodents can be up-and down regulated by different internal and external stimuli. Physical exercise in a running wheel being among the factors that have been most investigated. Since voluntary exercise not only increases adult neurogenesis in the hippocampus but also beneficially affects learning and memory in laboratory mice and rats, a widespread assumption holds a direct relationship between AHN and cognitive brain health also in higher order species, including humans. However, translating findings in laboratory rodents to the human condition faces difficulties. Enormous differences in basal rates of adult neurogenesis have been reported between mammalian species. The low level of AHN in primates and the complete lack of adult neurogenesis in bat species indicate species-specific differences in adult neurogenesis not only on a regulatory but also on a functional level. For a better understanding of species-specific differences in the regulation of AHN, we investigated basal rates of adult neurogenesis in laboratory mice and closely related wild mouse strains as well as the reaction of AHN to motivationally different running conditions. Testing different wild- and laboratory mice in the same environment allowed the identification of species-specific differences as well as possible domestication effects. Basal rates of adult hippocampal neurogenesis in equally-aged and genetically identical laboratory C57BL/6 mice show individual differences possibly reflecting epigenetic factors. However, the initial level of adult neurogenesis does not influence the response to wheel-exercise. Voluntary physical exercise in laboratory mice always increases AHN but this positive effect cannot be additively stimulated by enhanced running and is even lost as soon as the mice are forced to run. Rewarding the mice for their performance leads to an increase in wheel activity but does not translate into a corresponding additive increase in adult neurogenesis. Likewise, a more naturalistic situation, in which laboratory mice must run to obtain their daily food does not lead to an increase in cell proliferation and entails only a small increase in the number of young neurons, far below the one in voluntary running mice. Wild wood mice (Apodemus sylvaticus) and wild-derived western house mice (Mus musculus domesticus), both close relatives of the common laboratory mouse strains, were tested in the same running situations as laboratory C57BL/6 mice. Besides species-differences in basal neurogenesis rate, we find adult neurogenesis in wild mice remaining relatively constant in response to external influences. None of the factors that normally affect AHN in laboratory animals, such as stress, environmental changes or physical exercise, have an effect on adult neurogenesis in these animals. In wood mice, neither voluntary wheel running nor stress or an impoverished cage environment affect the number of newly generated neurons. House mice also show a stable adult neurogenesis, which shows no significant change after voluntary running or running for food. Adult neurogenesis in the dentate gyrus is thus regulated differently in laboratory and wild mice. However, even in laboratory mice it is not as plastic as initially suggested: laboratory mice, which are tested in a more naturalistic and complex running situation, show rather weak plasticity of AHN, resembling wild mice. Hence, it seems that the regulatory difference in adult neurogenesis between laboratory- and wild mice is, that laboratory animals react to a single stimulus in absence of other inputs. We believe that the constant exposure to different stimuli potentially affecting AHN has led to a natural selection that stabilizes adult neurogenesis in the wild. In contrast, during domestication - including inbreeding - much of the homeostatic capacity in regulating adult neurogenesis might have been lost. Taken together, our data imply that genetic (species-specific differences as well as within-species variation) play an important role in determining basal rates of adult neurogenesis, while motivational-contextual factors modulate the response of AHN to physical exercise, albeit chiefly in domesticated laboratory strains . As such differences appear already between phylogenetically closely related species, extrapolating findings in laboratory mice to distantly related taxonomic groups, such as humans, obviously requires much caution.

Objective To investigate whether mild hypothermia can promote neurogenesis in the dentate gyrus of hippocampus and cognitive function recovery after traumatic brain injury (TBI) through inhibiting apoptosis of hippocampal neurons. Methods A total of 66 healthy adult Sprague-Dawley rats were randomly divided into sham group, TBI group and TBI+ hypothermia group, with 22 rats in each group. The rat TBI model was established using the fluid percussion device. The rats in TBI+ hypothermia group received 4-hour hypothermia therapy immediately after injury, with the target temperature of 33.5℃. Bromodeoxyuridine (BrdU) was injected into the rats' abdominal cavity to label the mitotic cells. The test of Morris water maze was used to evaluate the rats' spatial learning and memory capabilities. Immunofluorescence staining was used to observe the expression levels of BrdU, doublecortin (DCX), neuron specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3) and cleaved caspase-3 expressions in dentate gyrus of hippocampus at 7 days and 28 days after injury. Expressions apoptosis-related proteins including the factor associated suicide (FAS)/factor associated suicide ligand (FASL), B-cell lymphoma-2 (Bcl-2), caspase-3 and cleaved caspase-3 expressions were detected by Western blot assay. Results The water maze tests at 28 days after injury showed that compared with TBI group, the escape latency in TBI+ hypothermia group was significantly shorter [(24.2±5.9)s∶(18±4.1)s], and both the time in the target quadrant and the number of platform crossing were increased significantly [(24.9±6.5)s∶(31.7±5.2)s; (1.9±0.8) times∶(3.5±1.2)times](P<0.05). Compared with the sham group, in TBI group and TBI+ hypothermia group, the BrdU+ new-born cells in the dentate gyrus of hippocampus were significantly increased at 7 days after injury [(9.4±4.1)∶(33.4±3.8); (9.4±4.1)∶(45.8±5.6)], the BrdU+ /DCX+ new-born neurons were increased at 7 days after injury [(2.0±0.6)∶(9.6±1.6); (2.0±0.6)∶(19.2±3.7)], and the BrdU+ /NeuN+ mature neurons were increased at 28 days after injury [(2.6±1.0)∶(17.2±3.9); (2.6±1.0)∶(33.6±9.1)] (P<0.01). TBI group showed more obvious increase than the TBI+ hypothermia group (P<0.01). Moreover, compared with 7 days after injury, the number of BrdU+ cells at 28 days after injury was further increased in TBI+ hypothermia group but decreased in TBI group [(45.8±5.6)∶(58.8±9.2); (33.4±3.8)∶(22.0±3.5)](P<0.05 or <0.01). Compared with the sham group, the caspase-3+ NeuN+ and caspase-3+ NeuN+ apoptotic neurons were significantly increased at 7 days after injury in TBI group [(2.0±0.9)∶(11.6±2.6); (2.6±1.0)∶(10.2±2.9)] (P<0.05). Compared with the TBI group, the cleaved caspase-3+ NeuN+ apoptotic neurons were decreased in TBI+ hypothermia group [(6.6±2.0)∶(11.6±2.6)](P<0.05). Furthermore, compared with the TBI group, mild hypothermia might down-regulate the expression of FAS, FASL, cleaved caspase-3 and caspase-3 and up-regulate the expression of Bcl-2 in the hippocampus [(1.54±0.15) ∶(1.14±0.12); (1.06±0.04)∶(0.80±0.09); (0.84±0.03)∶(0.62±0.08); (0.93±0.06)∶(0.86±0.09); (0.71±0.01)∶(1.58±0.18)](P<0.05). Conclusions Mild hypothermia might inhibit apoptosis of hippocampal neurons through cleaved caspase-3, FAS/FASL and Bcl-2 pathways, thus improving the neurogenesis and maturation of neurons in the dentate gyrus of hippocampus and facilitating cognitive function recovery in rats. It indicates that the function of hypothermia in anti-apoptosis and neurogenesis and maturity of hippocampal neurons may have a potential role in predicting the prognosis of TBI patients. Key words: Hypothermia; Brain injuries; Hippocampus; Neuve regeneration; Apoptosis

New neurons are generated throughout life. This discovery has challenged firmly held concepts about the structural plasticity and regenerative capacity of the mammalian brain. In this special issue of the EJN, leaders in the field summarize and review recent advances aiming to understand the molecular mechanisms underlying adult neurogenesis and the impact of new neurons on brain function in health and disease. Below we discuss pivotal yet unsolved aspects of adult neurogenesis as well as potential future directions in the field.

Objective To investigate the effects of isoflurane and sevoflurane at the equivalent depth of subanesthesia on neuronal proliferation and phosphorylation of extraceullar signal-regulated kinase 1/2 (ERK1/2) protein in the hippocampi of neonatal rats. Methods Seventy-two neonatal rats at postnatal day 7 were involved in this study and they were assigned randomly into isoflurane group (Iso group), sevoflurane group (Sev group) and control group (Con group). The rats in I group, S group or C group were separately exposed to 0.75% isoflurane or 1.2% sevoflurane (equivalent to 0.3 MAC for neonatal rats) or air for 6 h. Some rats in each group were injected intraperitoneally BrdU 100 mg/kg immediately (D0) (n=6) or 3 days after exposure (D3) (n=6), and their brains were perfusion and embedded by paraffin 24 h after BrdU injection. BrdU positive expressions in the in dentate gyrus (DG) area of hippocampus were detected by IHC staining. Besides, the fresh hippocampi of some rats each group were dissected at the end of anesthesia, caspase-3 and phospho-ERK1/2, ERK1/2 proteins expression were detected by Western blot (n=6). The other rats in each group were used to measure changes of pH and blood glucose (n=6). One way ANOVA test was used for data analysis among groups. Results BrdU-positive cells had no significant difference among group IsoD0((1332.43±192.70)/mm2), group SevD0((1207.33±139.50)/mm2), and group ConD0((1362.40±227.90)/mm2) at D0, while which had significantly decreased by 32.6% (P<0.05) in group IsoD3((604.56±65.77)/mm2) when compared with those in group ConD3((896.90±78.77)/mm2) at D3. There was no significant difference between groups of SevD3((808.73±41.27)/mm2) and ConD3. The expression of caspase-3 protein was increased by 195% (P<0.01) in Iso group while which only increased by 74% (P<0.05) in Sev group when compared with Con group. The expression of P44 and P42 of phospho-ERK1/2 protein in the hippocami decreased by 53% (P<0.01) and 47% (P<0.01)) seperately in Iso group when compared with Con group, while there were no significant differences between Sev group and Con group. Conclusion 0.3 MAC isoflurane, not sevoflurane inhibits neuronal proliferation in DG of hippocampi in the neonatal rats. Inhibiting ERK1/2 phosphorylation may involve in the mechanisms of that isoflurane inhibits neuronal proliferation. Key words: Isoflurane; Sevoflurane; Hippocampus; Neuronal proliferation

Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) play a critical role in neurogenesis. In the present study, we evaluated the additive effect of administering these two factors on post-ischemic progenitor cell proliferation, survival, and phenotypic maturation in the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) in the adult rat brain after transient middle cerebral artery occlusion. A combination of EGF+FGF-2 (each 1.44 ng/d) was continuously administered into the lateral ventricles for 3 days, 5-bromodeoxyuridine (BrdUrd) was injected (50 mg/Kg) twice daily for 3 days starting on Day 1 of reperfusion, and cohorts of rats were sacrificed on Day 5 and Day 21 of reperfusion. Compared with sham controls, ischemic rats showed a significantly higher number of newly proliferated cells in both the DG (by 766 +/- 37%, P < 0.05) and the SVZ (by 650 +/- 43%, P < 0.05). Of the progenitor cells proliferated on Day 5 after ischemia, 41 +/- 6% in the DG and 28 +/- 5% in the SVZ survived to 3 weeks. Compared with vehicle control, the EGF + FGF-2 infusion significantly increased the post-ischemic progenitor cell proliferation (by 319 +/- 40%, P < 0.05 in the DG and by 366 +/- 32%, P < 0.05 in the SVZ) and survival (by 40 +/- 12%, P < 0.05 in the DG and by 522 +/- 47%, P < 0.05 in the SVZ) studied at 5 and 21 days, respectively. Furthermore, of the newly proliferated cells survived to 3 weeks after ischemia, EGF + FGF-2 infusion caused a significantly higher number of neuronal nuclear protein-BrdUrd double-positive mature neurons in the DG (46 +/- 9%, P < 0.05) compared with vehicle control. Neuronal nuclear protein and BrdUrd double-positive mature neurons were also found in the DG. Glial fibrillary acidic protein-positive astrocytes did not show double-positive staining in either region. Specific growth factor infusion enhances post-ischemic progenitor cell proliferation by 5 days of reperfusion and neuronal maturation by 21 days of reperfusion in both the DG and SVZ in the adult rat brain.

The adult mammalian brain can produce new neurons in a process called adult neurogenesis, which occurs mainly in the subventricular zone (SVZ) and in the hippocampal dentate gyrus (DG). Brain-derived neurotrophic factor (BDNF) signaling and cannabinoid type 1 and 2 receptors (CB1R and CB2R) have been shown to independently modulate neurogenesis, but how they may interact is unknown. We now used SVZ and DG neurosphere cultures from early (P1-3) postnatal rats to study the CB1R and CB2R crosstalk with BDNF in modulating neurogenesis. BDNF promoted an increase in SVZ and DG stemness and cell proliferation, an effect blocked by a CB2R selective antagonist. CB2R selective activation promoted an increase in DG multipotency, which was inhibited by the presence of a BDNF scavenger. CB1R activation induced an increase in SVZ and DG cell proliferation, being both effects dependent on BDNF. Furthermore, SVZ and DG neuronal differentiation was facilitated by CB1R and/or CB2R activation and this effect was blocked by sequestering endogenous BDNF. Conversely, BDNF promoted neuronal differentiation, an effect abrogated in SVZ cells by CB1R or CB2R blockade while in DG cells was inhibited by CB2R blockade. We conclude that endogenous BDNF is crucial for the cannabinoid-mediated effects on SVZ and DG neurogenesis. On the other hand, cannabinoid receptor signaling is also determinant for BDNF actions upon neurogenesis. These findings provide support for an interaction between BDNF and endocannabinoid signaling to control neurogenesis at distinct levels, further contributing to highlight novel mechanisms in the emerging field of brain repair.

Stroke is caused by occlusion of a cerebral artery, which gives rise to focal ischemia with irreversible injury in a core region and partially reversible damage in the surrounding penumbra zone. In another type of insult, abrupt and near-total interruption of cerebral blood flow as a consequence of cardiac arrest or coronary artery occlusion leads to global ischemia and selective death of certain vulnerable neuronal populations such as the pyramidal neurons of hippocampal CA1. During the last decade, these ischemic insults have been reported to induce the formation of new neurons in the adult rodent brain from neural stem cells (NSCs) located in two regions: the subventricular zone (SVZ), lining the lateral ventricle, and the subgranular zone (SGZ) in the dentate gyrus (DG). Ischemia-induced neurogenesis is triggered both in areas where new neurons are normally formed, such as the DG, and in areas that are nonneurogenic in the intact brain, e.g., the striatum. These findings have raised several important issues: (1) Is the evidence for the formation of new neurons really solid or could there be other interpretations such as aberrant DNA synthesis caused by the ischemic insult in already existing, mature neurons? (2) What are the functional consequences of ischemia-induced neurogenesis? (3) Because the neurogenic response is minor and recovery after stroke incomplete, how can this presumed self-repair mechanism be boosted? In this chapter, we summarize the current status of research on neurogenesis after stroke. We also discuss the basic scientific problems that need to be addressed before this...

Objective To observe neural stem cells proliferation, migration and differentiation in hippocampus in developing rats with status epileptictus. Methods 320 healthy SD rats at age 7, 14, 21, 28 d (P7, P14, P21, P28) were randomly divided into status epilepticus (SE) and normal control group. In each group those rats at the same age were further randomly divided into 1, 7, 14, 21, 28 d five time points after PTZ-induced SE (n=8). New cell proliferation and migration were observed by immunohistochemistry studies in the dentate gyrus. Double labeling with Brdu/NeuN and Brdu/GFAP was performed in the P14 rats. Results Nestin positive cells appeared in the dentate gyrus on 1 d after SE in P7, P14, P21, P28 rats. The number of nestin positive cells gradually increased on 7 d and reached a peak on 14 d, then gradually reduced on 21 d, finally fell to a minimum on 28 d after SE. The numbers of nestin positive cells on 7 d（177.00±3.22，t=16.033）and 14 d （195.00±3.41，t=28.840） were significantly higher in the SE group than the NS group (147.50±2.08，136.50±2.65，both P＜0.05). The smaller age of rats with SE onset, the greater the nestin intensity. But the number of nestin positive cells in the dentate gyrus of normal rats were gradually decreased with increasing age. Nestin positive cells were distributed in subgranular zone of dentate gyrus on 1 d and 7 d after SE, then gradually migrated to the granule cell layer on 14 d with morphological changes. Small part of nestin positive cells were ectopically migrated to the hilus of dentate gyrus in P14, P21, P28 age rats, and were also seen in the CA1，CA3 of hippocampus and cortex with various cell morphology. For differentiation of newly generated cells, most of Brdu positive cells co-expressed NeuN and about 4%—5% cells co-expressed GFAP. Conclusions SE could induce neurogenesis in the hippocampal dentate gyrus area in developing rats which has age-related characteristics. Most new cells migrate from the subgranular zone to the granule cell layer of the dentate gyrus, and a small number of newly generated cells ectopically migrated to the hilus of dentate. The majority of newly generated cells differentiate into neurons, and the others differentiate into glial. Key words: Status epilepticus; Hippocampus; Neurogenesis; Neural stem cells

To explore the effects of proBDNF on cell proliferation and differentiation in hippocampal dentate gyrus in Alzheimer' disease (AD) rat model. The AD rat model was established. Alzet osmotic minipumps were connected to right hippocampus of AD rat and filled with proBDNF, sheep antibody to proBDNF or normal sheep serum respectively. Rats received the injection for 14 days at the speed of 0.5 microl/h. 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg, ip) was injected twice daily for 14 days. BrdU immunohistochemistry was processed to determine the number of newly generated cells. To examine the phenotype of newly generated cells, immunofluorescent triple labeling was conducted to colocalize BrdU-positive cells with rabbit anti-doublecortin (DCX) or mouse anti-glial fibrillary acid protein (GFAP). proBDNF group had fewer BrdU positive cells in dentate gyrus (P < 0.01), while anti-proBDNF group had more BrdU positive cells (P < 0.01) as compared with control group respectively. Immunofluorescent triple labeling showed that there was no phenotypic difference of BrdU positive cells between each group. proBDNF can suppress the proliferation of hippocampal neuron in dentate gyrus in AD rats while anti-proBDNF has the opposite effect. These findings suggest that promoting the hippocampal neurogenesis by blocking the functions of endogenous proBDNF may be a potential therapeutic strategy for AD.

During the development of the dentate gyrus (DG), both at embryonic and postnatal stages, radial glial cells (RGCs) and neural progenitors proliferate and generate mature granule neurons, the principal neuron of the DG. In an unique way, in the adult DG, a subpopulation of progenitors with a radial morphology are retained in a quiescent state as adult radial glial-like cells (RGLs) in the subgranular zone (SGZ) of the DG and continue to produce new granule neurons throughout adult life. This raises questions about when and how adult RGLs are generated in the DG, which are essential questions to understand how neurogenic niches are generated and maintained in the adult brain. HMG-box transcription factors of Sox family genes could be at the core of those processes, as many of them have essential regulatory functions in both developmental and adult neurogenesis. In this study, we have focused on SoxD transcription factors (Sox5 and Sox6) in DG neurogenesis, as our laboratory has previously shown that they play a critical role in regulating cell cycle progression in progenitor cells and that they are expressed in the SGZ, both during DG development and in adulthood. We describe now that during DG development both Sox5 and Sox6 are persistently expressed in RGCs/RGLs and that their expression gradually turns off along the progression of those cells towards the neuronal lineage. By conditional deletion of Sox5 and Sox6 from early central nervous system development, we have determined that Sox5 is required for RGCs/RGLs to enter the quiescent state during postnatal development. Thus, deleting Sox5 expression during development results first in an increase in hippocampal neurogenesis in young adults and then, in mature adults, leads to an exhaustion of RGLs pool. Furthermore, we have found that BMP signaling target, Id2, could be mediating Sox5-regulated quiescence acquisition during DG development. Furthermore, we have found that Sox6 alone is less required than Sox5 for the development of DG, at least during first three postnatal weeks. Interestingly, selective creERT/tamoxifen-induced deletion of SoxD genes in the adult DG, showed that both Sox5 and Sox6 are required for RGLs to transit from quiescent into active proliferative states, and consequently they are needed for adult neurogenesis. Taken together, our results prove that the transition from developmental RGCs into adult RGLs during DG development is regulated by Sox5. These results set up the basis to further explore Sox5 direct targets to understand the molecular mechanism that involve how adult neurogenesis is specifically generated at certain brain areas and how could we modulate the neurogenic process in pathological and ageing brains.