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The mechanisms underlying sexual differentiation of behavior and physiology in mammals and birds: relative contributions of sex steroids and sex chromosomes.
Front Neurosci
PUBLISHED: 08-14-2014
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From a classical viewpoint, sex-specific behavior and physiological functions as well as the brain structures of mammals such as rats and mice, have been thought to be influenced by perinatal sex steroids secreted by the gonads. Sex steroids have also been thought to affect the differentiation of the sex-typical behavior of a few members of the avian order Galliformes, including the Japanese quail and chickens, during their development in ovo. However, recent mammalian studies that focused on the artificial shuffling or knockout of the sex-determining gene, Sry, have revealed that sex chromosomal effects may be associated with particular types of sex-linked differences such as aggression levels, social interaction, and autoimmune diseases, independently of sex steroid-mediated effects. In addition, studies on naturally occurring, rare phenomena such as gynandromorphic birds and experimentally constructed chimeras in which the composition of sex chromosomes in the brain differs from that in the other parts of the body, indicated that sex chromosomes play certain direct roles in the sex-specific differentiation of the gonads and the brain. In this article, we review the relative contributions of sex steroids and sex chromosomes in the determination of brain functions related to sexual behavior and reproductive physiology in mammals and birds.
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Involvement of hemeoxygenase-1 in di(2-ethylhexyl) phthalate (DEHP)-induced apoptosis of Neuro-2a cells.
J Toxicol Sci
PUBLISHED: 03-21-2014
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A widely-used plasticizer di(2-ethylhexyl) phthalate (DEHP) is known to induce apoptosis in neurons, although the mechanisms responsible for DEHP-induced apoptosis is not well explored yet. We recently showed that exposure to DEHP increases the expression of hemeoxygenase (HO)-1, an oxidative stress related enzyme, in the mice brain. In this study, we investigated whether HO-1 is involved in DEHP-induced apoptosis using a mouse neuroblastoma cell line Neuro-2a, which forcibly express SCAT3, a fluorescent indicator of caspase-3 activity. The doses of DEHP at 1, 10 or 100 µM were used in the present study to mimic the level of human exposure to DEHP. Live image analysis of SCAT3-expressing Neuro-2a cells revealed that caspase-3 activity in the cells was significantly increased by DEHP at 100 µM but not 1 or 10 µM. We measured HO-1 mRNA level in Neuro-2a cells exposed to DEHP and found significant increase in HO-1 mRNA level by DEHP at 100 µM but not 1 or 10 µM. Live image analysis of SCAT3-expresisng Neuro-2a cells was further performed to determine the effects of HO-1 siRNA in DEHP-induced apoptosis via caspase-3 activation. We found that knockdown of HO-1 gene nullifies the effects of DEHP to activate caspase-3. These results suggest that HO-1 is involved in DEHP-induced apoptosis. Moreover, this study demonstrates that high-dose DEHP exposure induces caspase-3-dependent apoptosis, which is at least partially mediated by the up-regulation of HO-1 gene, in Neuro-2a cells.
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Regional difference in sex steroid action on formation of morphological sex differences in the anteroventral periventricular nucleus and principal nucleus of the bed nucleus of the stria terminalis.
PLoS ONE
PUBLISHED: 01-01-2014
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Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-? (ER?), but not estrogen receptor-? (ER?). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ER?, or ER? genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ER? genes, but not the ER? gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ER?, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ER? mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ER? mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ER? during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.
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Neuroanatomy and sex differences of the lordosis-inhibiting system in the lateral septum.
Front Neurosci
PUBLISHED: 01-01-2014
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Female sexual behavior in rodents, termed lordosis, is controlled by facilitatory and inhibitory systems in the brain. It has been well demonstrated that a neural pathway from the ventromedial hypothalamic nucleus (VMN) to the midbrain central gray (MCG) is essential for facilitatory regulation of lordosis. The neural pathway from the arcuate nucleus to the VMN, via the medial preoptic nucleus, in female rats mediates transient suppression of lordosis, until female sexual receptivity is induced. In addition to this pathway, other regions are involved in inhibitory regulation of lordosis in female rats. The lordosis-inhibiting systems exist not only in the female brain but also in the male brain. The systems contribute to suppression of heterotypical sexual behavior in male rats, although they have the potential ability to display lordosis. The lateral septum (LS) exerts an inhibitory influence on lordosis in both female and male rats. This review focuses on the neuroanatomy and sex differences of the lordosis-inhibiting system in the LS. The LS functionally and anatomically links to the MCG to exert suppression of lordosis. Neurons of the intermediate part of the LS (LSi) serve as lordosis-inhibiting neurons and project axons to the MCG. The LSi-MCG neural connection is sexually dimorphic, and formation of the male-like LSi-MCG neural connection is affected by aromatized testosterone originating from the testes in the postnatal period. The sexually dimorphic LSi-MCG neural connection may reflect the morphological basis of sex differences in the inhibitory regulation of lordosis in rats.
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Visualisation and characterisation of oestrogen receptor ?-positive neurons expressing green fluorescent protein under the control of the oestrogen receptor ? promoter.
Eur. J. Neurosci.
PUBLISHED: 03-21-2013
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Oestrogen receptor (ER)? plays important roles in the development and function of various neuronal systems through activation by its ligands, oestrogens. To visualise ER?-positive neurons, we generated transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the ER? promoter. In three independent tg lines, GFP-positive neurons were observed in areas previously reported to express ER? mRNA, including the lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus (MPO), hypothalamus, and amygdala. In these areas, GFP signals mostly overlapped with ER? immunoreactivity. GFP fluorescence was seen in neurites and cell bodies of neurons. In addition, the network and detailed structure of neurites were visible in dissociated and slice cultures of hypothalamic neurons. We examined the effect of oestrogen deprivation by ovariectomy on the structure of the GFP-positive neurons. The area of ER?-positive cell bodies in the bed nucleus of the stria terminalis and MPO was measured by capturing the GFP signal and was found to be significantly smaller in ovariectomy mice than in control mice. When neurons in the MPO were infected with an adeno-associated virus that expressed small hairpin RNA targeting the ER? gene, an apparent induction of GFP was observed in this area, suggesting a negative feedback mechanism in which ER? controls expression of the ER? gene itself. Thus, the ER? promoter-GFP tg mice will be useful to analyse the development and plastic changes of the structure of ER?-expressing neurons and oestrogen and its receptor-mediated neuronal responses.
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Collapsin response mediator protein 4 affects the number of tyrosine hydroxylase-immunoreactive neurons in the sexually dimorphic nucleus in female mice.
Dev Neurobiol
PUBLISHED: 02-12-2013
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In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV.
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Effects of sodium arsenite on neurite outgrowth and glutamate AMPA receptor expression in mouse cortical neurons.
Neurotoxicology
PUBLISHED: 02-10-2013
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There has been broad concern that arsenic in the environment exerts neurotoxicity. To determine the mechanism by which arsenic disrupts neuronal development, primary cultured neurons obtained from the cerebral cortex of mouse embryos were exposed to sodium arsenite (NaAsO2) at concentrations between 0 and 2 ?M from days 2 to 4 in vitro and cell survival, neurite outgrowth and expression of glutamate AMPA receptor subunits were assessed at day 4 in vitro. Cell survival was significantly decreased by exposure to 2 ?M NaAsO2, whereas 0.5 ?M NaAsO2 increased cell survival instead. The assessment of neurite outgrowth showed that total neurite length was significantly suppressed by 1 ?M and 2 ?M NaAsO2, indicating that the lower concentration of NaAsO2 impairs neuritogenesis before inducing cell death. Immunoblot analysis of AMPA receptor subunit expression showed that the protein level of GluA1, a specific subunit of the AMPA receptor, was significantly decreased by 1 ?M and 2 ?M NaAsO2. When immunocytochemistry was used to confirm this effect by staining for GluA1 expression in neuropeptide Y neurons, most of which contain GluA1, GluA1 expression in neuropeptide Y neurons was found to be significantly suppressed by 1 ?M and 2 ?M NaAsO2 but to be increased at the concentration of 0.5 ?M. Finally, to determine whether neurons could be rescued from the NaAsO2-induced impairment of neuritogenesis by compensatory overexpression of GluA1, we used primary cultures of neurons transfected with a plasmid vector to overexpress either GluA1 or GluA2, and the results showed that GluA1/2 overexpression protected against the deleterious effects of NaAsO2 on neurite outgrowth. These results suggest that the NaAsO2 concentration inducing neurite suppression is lower than the concentration that induces cell death and is the same as the concentration that suppresses GluA1 expression. Consequently, the suppression of GluA1 expression by NaAsO2 seems at least partly responsible for neurite suppression induced by NaAsO2.
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Effects of aromatase or estrogen receptor gene deletion on masculinization of the principal nucleus of the bed nucleus of the stria terminalis of mice.
Neuroendocrinology
PUBLISHED: 03-15-2011
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The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is a sexually dimorphic nucleus, and the male BNSTp is larger and has more neurons than the female BNSTp. To assess the roles of neuroestrogen synthesized from testicular androgen by brain aromatase in masculinization of the BNSTp, we performed morphometrical analyses of the adult BNSTp in aromatase knockout (ArKO), estrogen receptor-? knockout (?ERKO), and estrogen receptor-? knockout (?ERKO) mice and their respective wild-type littermates. In wild-type littermates, the BNSTp of males had a larger volume and greater numbers of neuronal and glial cells than did that of females. The volume and neuron number of the BNSTp in ArKO and ?ERKO males and glial cell number of the BNSTp in ?ERKO males were significantly smaller than those of wild-type male littermates, and they were not significantly different from those in female mice with either gene knockout. In contrast, there was no significant morphological difference in the BNSTp between ?ERKO and wild-type mice. Next, we examined the BNSTp of ArKO males subcutaneously injected with estradiol benzoate (EB) on postnatal days 1, 2, and 3 (1.5 ?g/day). EB-treated ArKO males had a significantly greater number of BNSTp neurons than did oil-treated ArKO males. The number of BNSTp neurons in EB-treated ArKO males was comparable to that in wild-type males. These findings suggested that masculinization of the BNSTp in mice involves the actions of neuroestrogen that was synthesized by aromatase and that this estrogen mostly binds to ER? during the postnatal period.
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Differential mRNA expression of neuroimmune markers in the hippocampus of infant mice following toluene exposure during brain developmental period.
J Appl Toxicol
PUBLISHED: 03-05-2011
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Toluene, a volatile organic compound with a wide range of industrial applications, can exert neurotoxic and immunotoxic effects. However, the effects of toluene exposure on developmental immunotoxicity in the brain have not yet been characterized. To investigate the susceptible window to toluene exposure during development and the effects of fetal and neonatal toluene exposure on the neuroimmune markers, gestational day (GD) 14 pregnant mice, postnatal day (PND) 2 and PND 8 male offspring were exposed to filtered air (control; 0?ppm), or 5 or 50?ppm toluene for 6?h per day for five consecutive days. The neuroimmune markers in the hippocampus of PND 21 were examined using a real-time RT-PCR and immunohistochemical analysis. Mice exposed to 50?ppm toluene on PND 2-6 showed significantly increased levels of nerve growth factor (NGF) and tumor necrosis factor (TNF)-? mRNAs. In contrast, NGF and brain-derived neurotrophic factor (BDNF) and proinflammatory cytokines TNF-?, CCL3, NF-?B, toll-like receptor (TLR)-4, astrocyte marker glial fibrillary acidic protein (GFAP), and microglia marker ionized calcium binding adapter molecule (Iba)-1 mRNAs were increased significantly in mice exposed to 5?ppm toluene on PND 8-12. These results indicate that low-level toluene exposure during the late postnatal period (PND 8-12) might induce neuroinflammatory mediators via TLR4-dependent NF-?B pathway in the hippocampus of PND 21 male mice. Among the three developmental phases, PND 8-12 seems to be most sensitive to toluene exposure. This is the first study to show developmental phase- and dose-specific changes in neuroimmune markers in infant mice following toluene exposure.
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Intrauterine position affects estrogen receptor ? expression in the ventromedial nucleus of the hypothalamus via promoter DNA methylation.
Endocrinology
PUBLISHED: 10-06-2010
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There is well-established evidence in many mammalian species for effects of the intrauterine position (IUP) (the sex-specific positioning of the embryo) on postnatal brain function and behavior. We found that the IUP affects estrogen receptor (ER)? expression in adult female rats in the ventrolateral region of the hypothalamic ventromedial nucleus (vlVMH), which is associated with sexual behavior. The ER? expression level in the vlVMH was higher in females that developed in utero between two male siblings (2M females) than in those that developed between female siblings (2F females). We also found that the cytosine methylation status across the ER? promoter in the vlVMH was affected by the IUP, with greater methylation in 2F females. These findings showed a negative correlation between ER? expression levels in the vlVMH and methylation frequency in the ER? promoter. This suggests that genomic methylation sustains the effect of the fetal IUP on ER? expression in the vlVMH.
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Effect of D-cycloserine on spatial learning performance and memory function-related gene expression in mice following toluene exposure.
J. UOEH
PUBLISHED: 06-17-2010
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D-cycloserine (DCS), a partial N-methyl-D-aspartate (NMDA) receptor agonist, is a well-known cognitive enhancer. To investigate the effect of DCS in cognitive function following toluene exposure, eight-week-old male C3H/HeN mice were exposed to filtered air (0 ppm) or 50 ppm toluene for 6 h a day on 5 consecutive days a week for 6 weeks. The day after the day of final exposure, a spatial learning task was performed using a Morris water maze apparatus. During the learning task, some mice were treated with DCS intraperitoneally (20 mg/kg) 30 min before the first trial of the acquisition phase and probe trial. After completion of the spatial learning task, the hippocampus was collected from each mouse to examine memory function-related gene expression using the real-time RT-PCR method. During the acquisition phase, on day 3 and 4, toluene-exposed mice with DCS treatment showed significantly better learning performance than corresponding saline treated groups. Moreover, the toluene-exposed mice with DCS treatment also showed significantly improved memory retention during the probe trial and up-regulation of hippocampal NMDA receptor subunit 2B mRNA expression compared with the saline treated groups. Our findings indicate that a subunit-specific modulation of hippocampal NMDA receptor mRNA expression by DCS contributes to improvement of spatial learning performance in mice following toluene exposure.
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Does early life toluene exposure alter the expression of NMDA receptor subunits and signal transduction pathway in infant mouse hippocampus?
Neurotoxicology
PUBLISHED: 06-09-2010
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We aim to investigate the critical window of susceptibility to toluene exposure during brain development and the effects of fetal and neonatal toluene exposure on the expression of N-methyl-d-aspartate (NMDA) receptor subunits and related transduction pathway in infant mice hippocampus. Pregnant mice (GD 14), male offspring (postnatal day; PND 2) or PND 8 were exposed to either a filtered air control (0ppm), or 5, or 50ppm of toluene for 6h per day for 5 consecutive days. On PND 21, the expression levels of NMDA receptor subunits, cyclic AMP responsive element binding protein (CREB)-1, calcium/calmodulin-dependent protein kinase (CaMK)-IV, and apoptotic related genes (Bax, Bcl) mRNAs in the hippocampus were estimated using quantitative real-time RT-PCR and immunohistochemical analyses. NR2B, CaMKIV and CREB1 mRNAs increased significantly in the hippocampus of mice exposed to 50ppm toluene on PND 2-6. In contrast, almost all memory function-related gene mRNAs and proapoptotic and anti-apoptotic ratio increased significantly in mice exposed to 5 or 50ppm toluene on PND 8-12. However, mice exposed to toluene on GD 14-18 showed no significant change. Increased active caspase-3 immunoreactive cells were found in hippocampal CA1 area of PND 21 male mice exposed to 5ppm toluene during PND 8-12. Our results suggest that late postnatal period may be a vulnerable and critical period to toluene exposure. Then, we have also examined the effect of toluene exposure in brain development on learning ability in young adult mice and found that poor spatial learning performance in PND 49 male mice exposed to 5ppm toluene during critical period. This is the first study to show that the early toluene exposure induces persistent of the alteration of memory function-related genes in infant mice and memory deficit in later life via modulating the synaptic morphology and function.
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Use of live imaging analysis for evaluation of cytotoxic chemicals that induce apoptotic cell death.
Toxicol In Vitro
PUBLISHED: 03-03-2010
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We carried out live imaging of PC12 cells expressing SCAT3, a caspase-3 cleavage peptide sequence linking two fluorescent proteins, ECFP and Venus, which function respectively as the donor and acceptor for FRET. Live imaging of SCAT3-expressing cells was performed from 60 to 300 min after exposure to sodium arsenite (NaAsO(2): 0, 1, 5, or 10 ?M) was initiated. We then measured the emission ratio of ECFP to Venus to monitor the activity of caspase-3 and found that the ratio was temporally and dose-dependently increased by NaAsO(2). The mean ECFP/Venus emission ratio between 200 and 300 min after exposure to NaAsO(2) at a dose of 5 or 10 ?M, but not at 1 ?M, was significantly higher than that in the control group. We showed by other methods that NaAsO(2) significantly increased the amount and activity of mature caspase-3 and the amount of nucleosomes generated from DNA fragmentation, and decreased cell viability. However, methods other than live imaging required a longer time and higher doses of NaAsO(2) than did live imaging to detect significant effects. This result suggests that live imaging using SCAT3 is a useful method for the screening of chemical toxicities and for improving the efficiency of toxicity evaluation.
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Up-regulation of neurotrophin-related gene expression in mouse hippocampus following low-level toluene exposure.
Neurotoxicology
PUBLISHED: 05-08-2009
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To investigate the role of strain differences in sensitivity to low-level toluene exposure on neurotrophins and their receptor levels in the mouse hippocampus, 8-week-old male C3H/HeN, BALB/c and C57BL/10 mice were exposed to 0, 5, 50, or 500 ppm toluene for 6h per day, 5 days per week for 6 weeks in an inhalation chamber. We examined the expressions of neurotrophin-related genes and receptors in the mouse hippocampus using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tyrosine kinase (Trk) A, and TrkB mRNAs in the C3H/HeN mice hippocampus was significantly higher in the mice exposed to 500 ppm toluene. Among the three strains of mice, the C3H/HeN mice seemed to be sensitive to toluene exposure. To examine the combined effect of toluene exposure and allergic challenge, the C3H/HeN mice stimulated with ovalbumin were exposed to toluene. The allergy group of C3H/HeN mice showed significantly elevated level of NGF mRNA in the hippocampus following exposure to 50 ppm toluene. Then, we also examined the expression of transcription factor, dopamine markers and oxidative stress marker in the hippocampus of sensitive strain C3H/HeN mice and found that the expression of CREB1 mRNA was significantly increased at 50 ppm toluene. In immunohistochemical analysis, the density of the NGF-immunoreactive signal was significantly stronger in the hippocampal CA3 region of the C3H/HeN mice exposed to 500 ppm toluene in non-allergy group and 50 ppm in allergy group. Our results indicate that low-level toluene exposure may induce up-regulation of neurotrophin-related gene expression in the mouse hippocampus depending on the mouse strain and an allergic stimulation in sensitive strain may decrease the threshold for sensitivity at lower exposure level.
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Effects of maternal toluene exposure on testosterone levels in fetal rats.
Toxicol. Lett.
PUBLISHED: 04-07-2009
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The goal of our study was to determine if toluene affected the synthesis and secretion of testosterone in fetal rats. Dams were exposed to atmospheres that contained 0.09 ppm, 0.9 ppm or 9 ppm of toluene for 90 min/day from gestational days (GDs) 14.5 to 18.5 via nasal inhalation. Fetal plasma testosterone concentrations determined by enzyme immunoassay were significantly reduced on GD 18.5 after exposure to 0.9 and 9 ppm, but not to 0.09 ppm, of toluene in male, but not in female, fetuses. We measured, using real-time PCR methods, mRNA levels in fetal testes for several steroidogenic enzymes involved in testosterone synthesis and insulin-like 3 (Insl3), a maker of Leydig cell differentiation. The mRNA levels of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were significantly reduced after exposure to 0.9-ppm toluene. However, the mRNA levels of cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17alpha-hydroxylase/c17-20 lyase, 17beta-hydroxysteroid dehydrogenase, and Insl3 were not significantly altered by exposure to 0.9-ppm toluene. In addition, immunohistochemical analysis showed reduced 3beta-HSD-immunoreactive areas in the interstitial region of fetal testes after exposure to 0.9 and 9 ppm, but not 0.09 ppm, toluene. These findings indicate that toluene reduced the synthesis and secretion of testosterone in fetal testes from rats possibly as a consequence of reduced 3beta-HSD expression.
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Inhibition of neurite outgrowth and alteration of cytoskeletal gene expression by sodium arsenite.
Neurotoxicology
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Arsenic compounds that are often found in drinking water increase the risk of developmental brain disorders. In this study, we performed live imaging analyses of Neuro-2a cells expressing SCAT3, a caspase-3 cleavage peptide sequence linking two fluorescent proteins; enhanced cyan fluorescence protein (ECFP) and Venus, to determine whether sodium arsenite (NaAsO(2); 0, 1, 5, or 10 ?M) affects both neurite outgrowth and/or induces apoptosis with the same doses and in the same cell cultures. We observed that the area ratio of neurite to cell body in SCAT3-expressing cells was significantly reduced by 5 and 10 ?M NaAsO(2), but not by 1 ?M, although the emission ratio of ECFP to Venus, an endpoint of caspase-3 activity, was not changed. However, cytological assay using apoptotic and necrotic markers resulted in that apoptosis, but not necrosis, was significantly induced in Neuro-2a cells when NaAsO(2) exposure continued after the significant effects of NaAsO(2) on neurite outgrowth were found by live imaging. These results suggested that neurite outgrowth was suppressed by NaAsO(2) prior to NaAsO(2)-induced apoptosis. Next, we examined the effects of NaAsO(2) on cytoskeletal gene expression in Neuro-2a cells. NaAsO(2) increased the mRNA levels of the light and medium subunits of neurofilament and decreased the mRNA levels of tau and tubulin in a dose-dependent manner; no significant effect was found in the mRNA levels of the heavy subunit of neurofilament, microtubule-associated protein 2, or actin. The changes in cytoskeletal gene expression are likely responsible for the inhibitory effects of NaAsO(2) on neurite outgrowth.
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Involvement of postnatal apoptosis on sex difference in number of cells generated during late fetal period in the sexually dimorphic nucleus of the preoptic area in rats.
Neurosci. Lett.
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Postnatal apoptosis is involved in formation of the sex difference in neuron number of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in rats. In this study, we examined the origin of neurons that die with apoptosis on the postnatal period to exhibit the sex difference in neuron number of the SDN-POA. First, we measured the number of cells that were labeled with 5-bromo-2-deoxyuridine (BrdU) on embryonic day (ED) 17, ED18, and ED19 in the SDN-POA of rats on postnatal day (PD) 4 and PD8. The SDN-POA had many more cells labeled with BrdU on ED17 and ED18 than those on ED19. Significantly fewer cells labeled with BrdU on ED18 in the female SDN-POA from PD4 to PD8 resulted in a significant sex difference in the number at PD8. Next, combination analyses of BrdU-labeling and immunohistochemistry for single-stranded DNA (ssDNA), an apoptotic marker, were succeeded to investigate whether SDN-POA neurons generated during ED17-18 were removed by apoptosis. Many more ssDNA-immunoreactive cells that had been labeled with BrdU during ED17-18 were found in the SDN-POA of PD8 females, but few in the SDN-POA of PD8 males and PD4 females and males. These results suggest that the sex difference in the number of SDN-POA neurons generated during the late fetal period was caused by postnatal apoptosis.
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