Vitamin D3 Enriches Ceramide Content in Exosomes Released by Embryonic Hippocampal Cells.

The release of exosomes can lead to cell-cell communication. Nutrients such as vitamin D3 and sphingolipids have important roles in many cellular functions, including proliferation, differentiation, senescence, and cancer. However, the specific composition of sphingolipids in exosomes and their changes induced by vitamin D3 treatment have not been elucidated. Here, we initially observed neutral sphingomyelinase and vitamin D receptors in exosomes released from HN9.10 embryonic hippocampal cells. Using ultrafast liquid chromatography tandem mass spectrometry, we showed that exosomes are rich in sphingomyelin species compared to whole cells. To interrogate the possible functions of vitamin D3, we established the optimal conditions of cell treatment and we analyzed exosome composition. Vitamin D3 was identified as responsible for the vitamin D receptor loss, for the increase in neutral sphingomyelinase content and sphingomyelin changes. As a consequence, the generation of ceramide upon vitamin D3 treatment was evident. Incubation of the cells with neutral sphingomyelinase, or the same concentration of ceramide produced in exosomes was necessary and sufficient to stimulate embryonic hippocampal cell differentiation, as vitamin D3. This is the first time that exosome ceramide is interrogated for mediate the effect of vitamin D3 in inducing cell differentiation.

Defective Reelin/Dab1 signaling pathways associated with disturbed hippocampus development of homozygous yotari mice.

Homozygous Dab1 yotari mutant mice, Dab1yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways.

Mesenchymal stem cell­derived extracellular vesicles prevent neural stem cell hypoxia injury via promoting miR­210­3p expression.

Neural stem cells (NSCs) have the potential to give rise to offspring cells and hypoxic injury can impair the function of NSCs. The present study investigated the effects of mesenchymal stem cell (MSC)­derived extracellular vesicles (EVs) on NSC injury, as well as the underlying mechanisms. MSC­EVs were isolated and identified via morphological and particle size analysis. Cobalt chloride was used to establish a hypoxic injury model in NSCs. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was conducted to detect apoptosis. Reverse transcription­quantitative PCR was performed to detect the expression levels of miR­210­3p, and western blotting was used to detect the expression levels of apoptosis­inducing factor (AIF) and Bcl­2 19 kDa interacting protein (BNIP3). Compared with the control group, NSC apoptosis, and the expression of miR­210­3p, AIF and BNIP3 were significantly higher in the cobalt chloride­induced hypoxia group. By contrast, treatment with MSC­EVs further increased miR­210­3p expression levels, but reduced NSC apoptosis and the expression levels of AIF and BNIP3 compared with the model group (P<0.05). In addition, miR­210­3p inhibitor reduced miR­210­3p expression, but promoted hypoxia­induced apoptosis and the expression levels of AIF and BNIP3 compared with the model group (P<0.05). Collectively, the results suggested that MSC­EVs prevented NSC hypoxia injury by promoting miR­210­3p expression, which might reduce AIF and BNIP3 expression levels and NSC apoptosis.

Selection and identification of the panel of reference genes for quantitative real-time PCR normalization in rat testis at different development periods.

BACKGROUND: In quantitative real-time PCR (qRT-PCR), the expression levels of various adult reference genes may be unstable at different developmental periods and tissues, and will lead to inaccurate detected results. This study aimed to select and identify the optimal panel of reference genes in rat testis at different development periods. METHODS: We detected mRNA expression levels of five common rat testicular reference genes (GAPDH, ß-actin, 18s, RPS16 and RPL19) by qRT-PCR at different developmental periods (fetus, infancy, and adolescence), selected optimal panel of reference genes by combining with stability algorithms, and verified their tissue specificity. Lastly, we observed their mRNA expression alterations under pathological conditions to evaluate the stability and accuracy, and verify testicular dysplasia model. RESULTS: Based on comprehensive analysis, the best panel of reference genes of testis were GAPDH+ß-actin (at fetus) and GAPDH+RPL19 (at infancy and adolescent). Meanwhile, the best panel of reference genes of fetal testis was consistent with placenta and fetal hippocampus but different from fetal liver and kidney. Furthermore, in prenatal dexamethasone exposure (PDE) model, the results were consistent with those under physiological conditions, and the testicular steroidogenesis acute regulatory protein (StAR) was most obviously decreased when using the best panel of reference genes. CONCLUSION: In this study, rat testicular GAPDH+ß-actin for fetuses and GAPDH+RPL19 for infants and adolescents are recommended to be the optimal panel of reference genes. Respectively. The selected panel of reference genes was still stable under PDE condition. This study provided technical and theoretical supports for researches on testicular development toxicology.

Deletion of the Nucleotide Exchange Factor Vav3 Enhances Axonal Complexity and Synapse Formation but Tampers Activity of Hippocampal Neuronal Networks In Vitro.

Vav proteins activate GTPases of the RhoA subfamily that regulate the cytoskeleton and are involved in adhesion, migration, differentiation, polarity and the cell cycle. While the importance of RhoA GTPases for neuronal morphology is undisputed, their regulation is less well understood. In this perspective, we studied the consequences of the deletion of Vav2, Vav3 and Vav2 and 3 (Vav2-/-, Vav3-/-, Vav2-/-/3-/-) for the development of embryonic hippocampal neurons in vitro. Using an indirect co-culture system of hippocampal neurons with primary wild-type (WT) cortical astrocytes, we analysed axonal and dendritic parameters, structural synapse numbers and the spontaneous network activity via immunocytochemistry and multielectrode array analysis (MEA). Here, we observed a higher process complexity in Vav3-/-, but not in Vav2-/- neurons after three and five days in vitro (DIV). Furthermore, an enhanced synapse formation was observed in Vav3-/- after 14 days in culture. Remarkably, Vav2-/-/3-/- double knockout neurons did not display synergistic effects. Interestingly, these differences were transient and compensated after a cultivation period of 21 days. Network analysis revealed a diminished number of spontaneously occurring action potentials in Vav3-/- neurons after 21 DIV. Based on these results, it appears that Vav3 participates in key events of neuronal differentiation.

Hexagonal boron nitrides reduce the oxidative stress on cells.

The molecular stress caused by a drug administered to treat a disorder on healthy cells appears as a side effect. In this study, we aim to understand the potential of hexagonal boron nitrides (hBNs) as a therapeutic agent to relieve the cellular stress exerted by drugs. First, the cytotoxicity of hBNs and their possible degradation product, boric acid (BA), on the embryonic mouse hippocampal cell line mHippo E-14 was assessed in a wide concentration range (4.4-440 µg ml-1) of boron including hBNs and BA for 24 and 72 h exposure. Then, cell cycle, reactive oxygen species generation, cell death mechanism and apoptotic body formation in nuclei with hBN and BA exposure were evaluated at increased concentrations and incubation times. Finally, the cells, exposed to doxorubicin (DOX), an anti-cancer chemotherapy drug, to exert oxidative stress, were treated with hBNs and BA. The results indicate that hBNs decrease the oxidative stress at the concentrations that are nontoxic to cells. The study suggests that hBNs can open new venues for their investigation to reduce or eliminate the adverse effects of toxic drugs used in the treatment of several fatal diseases including neurological disorders and cancer with their slow degradation feature.

Decoding the development of the human hippocampus.

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and the consolidation of information from short-term memory to long-term memory1,2. Here we use single-cell RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis to illustrate the cell types, cell linage, molecular features and transcriptional regulation of the developing human hippocampus. Using the transcriptomes of 30,416 cells from the human hippocampus at gestational weeks 16-27, we identify 47 cell subtypes and their developmental trajectories. We also identify the migrating paths and cell lineages of PAX6+ and HOPX+ hippocampal progenitors, and regional markers of CA1, CA3 and dentate gyrus neurons. Multiomic data have uncovered transcriptional regulatory networks of the dentate gyrus marker PROX1. We also illustrate spatially specific gene expression in the developing human prefrontal cortex and hippocampus. The molecular features of the human hippocampus at gestational weeks 16-20 are similar to those of the mouse at postnatal days 0-5 and reveal gene expression differences between the two species. Transient expression of the primate-specific gene NBPF1 leads to a marked increase in PROX1+ cells in the mouse hippocampus. These data provides a blueprint for understanding human hippocampal development and a tool for investigating related diseases.

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice.

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.

Progress in iPSC-Based Modeling of Psychiatric Disorders.

Progress in iPSC-based cellular systems provides new insights into human brain development and early neurodevelopmental deviations in psychiatric disorders. Among these, studies on schizophrenia (SCZ) take a prominent role owing to its high heritability and multifarious evidence that it evolves from a genetically induced vulnerability in brain development. Recent iPSC studies on patients with SCZ indicate that functional impairments of neural progenitor cells (NPCs) in monolayer culture extend to brain organoids by disrupting neocorticogenesis in an in vitro model. In addition, the formation of hippocampal circuit-like structures in vitro is impaired in patients with SCZ as is the case for glia development. Intriguingly, chimeric-mice experiments show altered oligodendrocyte and astrocyte development in vivo that highlights the importance of cell-cell interactions in the pathogenesis of early-onset SCZ. Likewise, cortical imbalances in excitatory-inhibitory signaling may result from a cell-autonomous defect in cortical interneuron (cIN) development. Overall, these findings indicate that genetic risk in SCZ impacts neocorticogenesis, hippocampal circuit formation, and the development of distinct glial and neuronal subtypes. In light of this remarkable progress, we discuss current limitations and further steps necessary to harvest the full potential of iPSC-based investigations on psychiatric disorders.

Cajal-Retzius neurons are required for the development of the human hippocampal fissure.

Cajal-Retzius neurons (CRN) are the main source of Reelin in the marginal zone of the developing neocortex and hippocampus (HC). They also express the transcription factor p73 and are complemented by later-appearing GABAergic Reelin+ interneurons. The human dorsal HC forms at gestational week 10 (GW10), when it develops a rudimentary Ammonic plate and incipient dentate migration, although the dorsal hippocampal fissure (HF) remains shallow and contains few CRN. The dorsal HC transforms into the indusium griseum (IG), concurrently with the rostro-caudal appearance of the corpus callosum, by GW14-17. Dorsal and ventral HC merge at the site of the former caudal hem, which is located at the level of the future atrium of the lateral ventricle and closely connected with the choroid plexus. The ventral HC forms at GW11 in the temporal lobe. The ventral HF is wide open at GW14-16 and densely populated by large numbers of CRNs. These are in intimate contact with the meninges and meningeal blood vessels, suggesting signalling through diverse pathways. At GW17, the fissure deepens and begins to fuse, although it is still marked by p73/Reelin+ CRNs. The p73KO mouse illustrates the importance of p73 in CRN for HF formation. In the mutant, Tbr1/Reelin+ CRNs are born in the hem but do not leave it and subsequently disappear, so that the mutant cortex and HC lack CRN from the onset of corticogenesis. The HF is absent, which leads to profound architectonic alterations of the HC. To determine which p73 isoform is important for HF formation, isoform-specific TAp73- and DeltaNp73-deficient embryonic and early postnatal mice were examined. In both mutants, the number of CRNs was reduced, but each of their phenotypes was much milder than in the global p73KO mutant missing both isoforms. In the TAp73KO mice, the HF of the dorsal HC failed to form, but was present in the ventral HC. In the DeltaNp73KO mice, the HC had a mild patterning defect along with a shorter HF. Complex interactions between both isoforms in CRNs may contribute to their crucial activity in the developing brain.

Inhibition of protein tyrosine phosphatase 1B protects against sevoflurane-induced neurotoxicity mediated by ER stress in developing brain.

BACKGROUND: ER stress is involved in sevoflurane-induced neurotoxicity. Protein tyrosine phosphatase 1B (PTP1B) resided in the ER membrane is known to regulate ER stress. However, the role of PTP1B in sevoflurane-induced neurotoxicity is unknown. METHOD: Seven-day-old mice treated with 2.3% sevoflurane for 6 h as animal model. The hippocampal tissues were harvested following sevoflurane exposure for evaluation of ER stress markers with Western blot, ER morphology with transmission electron microscopy and density of dendrite spine with Golgi staining. In another subset of mice, neurocognitive function was assessed 4 weeks after anesthesia using Morris water maze test. We also examined the effects of PTP1B or PERK inhibitor on sevoflurane-induced neurodegeneration in the hippocampus. RESULT: The results showed inhibition of PTP1B significantly ameliorated sevoflurane-induced (1) ultrastructural ER alternations and ER stress activation as indicated by upregulation of PERK and eIF2α phosphorylation; (2) increase in cleaved caspase-3 expression; (3) elevated expressions of proinflammatory NF-κB and TNFα;(4) decreases in expression of synaptophysin, Arc and BDNF-TrkB proteins as well as loss of dendrite spine in the hippocampus; and (5) impairment in neurobehavioural performance at adolescence. Similarly, ER stress inhibitor is also found to downregulate eIF2α phosphorylation and neuroinflammation, and increase expressions of synaptic proteins and BDNF-TrkB signaling proteins. CONCLUSIONS: Our study shows PTP1B inhibition mitigates sevoflurane-induced neurodegeneration maybe mediated by ER stress in developing brain, and eventually improves cognitive function. This suggests PTP1B inhibition could represent a promising strategy to prevent sevoflurane-induced neurotoxicity.

New mechanisms of phenytoin in calcium homeostasis: competitive inhibition of CD38 in hippocampal cells.

PURPOSE: Phenytoin is a major anticonvulsant drug that is effective to improve arrhythmia and neuropathic pain. According to early works, phenytoin affected cell membrane depolarization by sodium channel blocking, guanylyl and adenylyl cyclase suppression that cause to intracellular Na+ and Ca2+ downregulation. This study was aimed to clarify some ambiguities in pathophysiological action of phenytoin by in vitro and molecular docking analyses. METHODS: In this study intracellular free Ca2+ of primary culture of embryonic mouse hippocampus evaluated via Fura 2 as fluorescent probe. The effects of phenytoin on ADP ribosyl cyclase activity was assessed by recently developed fluorometric assay. Molecular docking simulation was also implemented to investigate the possible interaction between phenytoin and CD38. RESULTS: Our results confirmed phenytoin competitively inhibits cyclase activity of CD38 (IC50 = 8.1 µM) and reduces cADPR content. cADPR is a Ca2+-mobilising second messenger which binds to L-type calcium channel and ryanodine receptors in cell and ER membrane and increases cytosolic free Ca2+. Ca2+ content of cells decreased significantly in the presence of phenytoin in a dose dependent manner (IC50 = 12.74 µM). Based on molecular docking analysis, phenytoin binds to deeper site of CD38 active site, mainly via hydrophobic interactions and consequently inhibits proper contact of substrate with catalytic residues specially Glu 226, Trp 186, Thr221. CONCLUSION: Taken together, one of the anticonvulsant mechanisms of phenytoin is Ca2+ inhibition from CD38 pathway, therefore could be used in disorders that accompanied by CD38 over production or activation such as heart disease, depression, brain sepsis, airway disease, oxidative stress and inflammation. Graphical abstract ᅟ.

NMDA receptors inhibit axonal outgrowth by inactivating Akt and activating GSK-3ß via calcineurin in cultured immature hippocampal neurons.

Neurons are highly polarized cells with an axon and dendritic arbors. It is still not well studied that how formation and elaboration of axon and dendrites is controlled by diffusible signaling factors such as glutamate via specific receptors. We found that N-methyl-D-aspartate (NMDA) receptors were enriched (stage 2-3) but decreased expression (stage 4-5) at tip of axon of cultured hippocampal neurons during distinct development stages. Inhibition of NMDA receptor activity by competitive antagonist DL-2-amino-5-phosphonovalerate (APV) or channel blocker MK801 promoted axonal outgrowth at the early stages, whereas inhibited dendritic development in later stages. Meanwhile, knockdown of NMDA receptors also promoted axonal outgrowth and branch in immature neurons. Furthermore, GluN2B but not GluN2A subunit inhibited axonal outgrowth in immature hippocampal neurons. Finally, we found that NMDA receptors inhibited axonal outgrowth by inactivating Akt and activating GSK-3ß signaling in a calcineurin-dependent manner. Taken together, our results demonstrate that stabilization GSK-3ß activation in the axon growth cone by Ca2+ influx through NMDA receptors may be involved in regulation of axon formation in immature neurons at early stages.

The signaling pathways underlying BDNF-induced Nrf2 hippocampal nuclear translocation involve ROS, RyR-Mediated Ca2+ signals, ERK and PI3K.

The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) induces complex neuronal signaling cascades that are critical for the cellular changes underlying synaptic plasticity. These pathways include activation of Ca2+ entry via N-methyl-D-aspartate receptors and sequential activation of nitric oxide synthase and NADPH oxidase, which via generation of reactive nitrogen/oxygen species stimulate Ca2+-induced Ca2+ release mediated by Ryanodine Receptor (RyR) channels. These sequential events underlie BDNF-induced spine remodeling and type-2 RyR up-regulation. In addition, BDNF induces the nuclear translocation of the transcription factor Nrf2, a master regulator of antioxidant protein expression that protects cells against the oxidative damage caused by injury and inflammation. To investigate the possible BDNF-induced signaling cascades that mediate Nrf2 nuclear translocation in primary hippocampal cultures, we tested here whether reactive oxygen species, RyR-mediated Ca2+ release, ERK or PI3K contribute to this response. We found that pre-incubation of cultures with inhibitory ryanodine to suppress RyR-mediated Ca2+ release, with the reducing agent N-acetylcysteine or with inhibitors of ERK or PI3K activity, prevented the nuclear translocation of Nrf2 induced by incubation for 6 h with BFNF. Based on these combined results, we propose that the key role played by BDNF as an inducer of neuronal antioxidant responses, characterized by BDNF-induced Nfr2 nuclear translocation, entails crosstalk between reactive oxygen species and RyR-mediated Ca2+ release, and the participation of ERK and PI3K activities.

Kisspeptin increases intracellular calcium concentration by protein kinase C-mediated signaling in the primary cultured neurons from rat hippocampus.

In addition to the fact that kisspeptin and its receptor GPR54 are well known to be abundantly expressed in the hypothalamus with suggestive roles in the initiation of puberty and similar reproductive system properties, there is also proof showing that kisspeptin might have influences on hippocampal functions. In our previous study, it was shown that kisspeptin increased free intracellular Ca2+ values ([Ca2+]i) through protein kinase C (PKC) activation in GT1-7 cells. For this reason, we examined the influences of kisspeptin on [Ca2+]i in hippocampal neurons to determine if kisspeptin shows its effects on hippocampus through the same mechanism. Hippocampal neurons were excised from the brains of fetuses on 17th embryonic day from maternal rats. The influences of kisspeptin on [Ca2+]i in hippocampal neurons were examined through in vitro calcium imaging system. The responses of [Ca2+]i to kisspeptin were quantified by the changes in 340nm/380nm ratio.  Kisspeptin-10 caused [Ca2+]i transients in hippocampal neurons. The change in [Ca2+]i by 100 nM kisspeptin was prevented by pre-treating the cells in PKC inhibitor chelerythrine chloride. According to the results, kisspeptin activates intracellular calcium signaling in hippocampal neurons via the pathway that depends on PKC. The results of this study suggest that kisspeptin may have a role in hippocampal neuron functions.

Midazolam Attenuates Autophagy and Apoptosis Caused by Ketamine by Decreasing Reactive Oxygen Species in the Hippocampus of Fetal Rats.

Every year between 0.75% and 2% of pregnant women require surgery that is related to either the pregnancy or other medical problems in USA. Therefore, the neurodegeneration following anesthesia in a variety of animal models has attracted our attention. Neurotoxic effects of ketamine cannot be ignored. In contrast, some anesthetics, including midazolam, protect neurons and increase dendritic spine density. However, the mechanism of neuroprotection by midazolam is not clear, and whether midazolam can relieve the damage caused by ketamine is unknown. Therefore, in this study, we explored the effects of midazolam on ketamine anesthesia. We measured protein levels of cleaved-caspase-3 (c-caspase-3), beclin-1, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), autophagy-related gene 4 (ATG4), ATG5, p62 (SQSTM1), and the autophagy marker light chain 3 (LC3) in hippocampus by Western analysis. We also measured total antioxidant capacity (T-AOC), and levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in hippocampus and PC12 cells. Results showed that ketamine induced apoptosis through activation of the mitochondrial pathway by increasing the expression of c-caspase-3 and Bax, and decreasing the expression of Bcl-2 at the protein level. Ketamine also increased the expression of LC3II and ATG5, proteins, decreased the expression of ATG4 and P62, and finally induced autophagy. Ketamine promoted the production of ROS and MDA, and reduced total antioxidant capacity (T-AOC); these effects were attenuated by midazolam. In conclusion, ketamine induces toxicity in human neurons through ROS-mediated activation of mitochondrial apoptotic pathway and autophagy. The harmful effects of ketamine can be ameliorated by midazolam.

Mid-gestational sevoflurane exposure inhibits fetal neural stem cell proliferation and impairs postnatal learning and memory function in a dose-dependent manner.

Advancements in fetal intervention procedures have led to increases in the number of pregnant women undergoing general anesthesia during the second trimester-a period characterized by extensive proliferation of fetal neural stem cells (NSCs). However, few studies have investigated the effects of mid-gestational sevoflurane exposure on fetal NSC proliferation or postnatal learning and memory function. In the present study, pregnant rats were randomly assigned to a control group (C group), a low sevoflurane concentration group (2%; L group), a high sevoflurane concentration group (3.5%; H group), a high sevoflurane concentration plus lithium chloride group (H + Li group), and a lithium chloride group (Li group) at gestational day 14. Rats received different concentrations of sevoflurane anesthesia for 2 h. The offspring rats were weaned at 28 days for behavioral testing (i.e., Morris Water Maze [MWM]), and fetal brains or postnatal hippocampal tissues were harvested for immunofluorescence staining, real-time PCR, and Western blotting analyses in order to determine the effect of sevoflurane exposure on NSC proliferation and the Wnt/ß-catenin signaling pathway. Our results indicated that maternal exposure to 3.5% sevoflurane (H group) during the mid-gestational period impaired the performance of offspring rats in the MWM test, reduced NSC proliferation, and increased protein levels of fetal glycogen synthase kinase-3 beta (GSK-3ß). Such treatment also decreased levels of ß-catenin protein, CD44 RNA, and Cyclin D1 RNA relative to those observed in the C group. However, these effects were transiently attenuated by treatment with lithium chloride. Conversely, maternal exposure to 2% sevoflurane (L group) did not influence NSC proliferation or the Wnt signaling pathway. Our results suggest that sevoflurane exposure during the second trimester inhibits fetal NSC proliferation via the Wnt/ß-catenin pathway and impairs postnatal learning and memory function in a dose-dependent manner.

Nestin-expressing cell types in the temporal lobe and hippocampus: Morphology, differentiation, and proliferative capacity.

Nestin is expressed in immature neuroepithelial and progenitor cell types and transiently upregulated in proliferative neuroglial cells responding to acute brain injury, including following seizures. In 36 temporal lobe (TLobe) specimens from patients with TLobe epilepsy (age range 8-60 years) we studied the number, distribution and morphology of nestin-expressing cells (NEC) in the pes, hippocampus body, parahippocampal gyrus, amygdala, temporal cortex and pole compared with post mortem control tissues from 26 cases (age range 12 gestational weeks to 76 years). The proliferative fraction of NEC was evaluated in selected regions, including recognized niches, using MCM2. Their differentiation was explored with neuronal (DCX, mushashi, ßIII tubulin, NeuN) and glial (GFAP, GFAPdelta, glutamine synthetase, aquaporin4, EAAT1) markers, both in sections or following culture. Findings were correlated with clinical parameters. A stereotypical pattern in the distribution and morphologies of NEC was observed, reminiscent of patterns in the developing brain, with increased densities in epilepsy than adult controls (p < .001). Findings included MCM2-positive radial glial-like cells in the periventricular white matter and rows of NEC in the hippocampal fimbria and sulcus. Nestin cells represented 29% of the hippocampal proliferative fraction in epilepsy cases; 20% co-expressed ßIII tubulin in culture compared with 28% with GFAP. Significant correlations were noted between age at surgery, memory deficits and nestin populations. TLobe NEC with ongoing proliferative capacity likely represent vestiges of developmental migratory streams and resident reactive cell populations of potential relevance to hippocampal epileptogenesis, TLobe pathology, and co-morbidities, including memory decline.

Aging and neurodegeneration are associated with increased mutations in single human neurons.

It has long been hypothesized that aging and neurodegeneration are associated with somatic mutation in neurons; however, methodological hurdles have prevented testing this hypothesis directly. We used single-cell whole-genome sequencing to perform genome-wide somatic single-nucleotide variant (sSNV) identification on DNA from 161 single neurons from the prefrontal cortex and hippocampus of 15 normal individuals (aged 4 months to 82 years), as well as 9 individuals affected by early-onset neurodegeneration due to genetic disorders of DNA repair (Cockayne syndrome and xeroderma pigmentosum). sSNVs increased approximately linearly with age in both areas (with a higher rate in hippocampus) and were more abundant in neurodegenerative disease. The accumulation of somatic mutations with age-which we term genosenium-shows age-related, region-related, and disease-related molecular signatures and may be important in other human age-associated conditions.

Polarity of varicosity initiation in central neuron mechanosensation.

Little is known about mechanical regulation of morphological and functional polarity of central neurons. In this study, we report that mechanical stress specifically induces varicosities in the axons but not the dendrites of central neurons by activating TRPV4, a Ca2+/Na+-permeable mechanosensitive channel. This process is unexpectedly rapid and reversible, consistent with the formation of axonal varicosities in vivo induced by mechanical impact in a mouse model of mild traumatic brain injury. In contrast, prolonged stimulation of glutamate receptors induces varicosities in dendrites but not in axons. We further show that axonal varicosities are induced by persistent Ca2+ increase, disassembled microtubules (MTs), and subsequently reversible disruption of axonal transport, and are regulated by stable tubulin-only polypeptide, an MT-associated protein. Finally, axonal varicosity initiation can trigger action potentials to antidromically propagate to the soma in retrograde signaling. Therefore, our study demonstrates a new feature of neuronal polarity: axons and dendrites preferentially respond to physical and chemical stresses, respectively.