Immature properties of large-conductance calcium-activated potassium channels in rat neuroepithelium

The pharmacological and biophysical properties of large-conductance Ca-activated K (BK) channels from embryonic rat telencephalic neuroepithelium were investigated with in situ patch-clamp techniques. A fraction of these channels exhibited properties characteristic of BK channels recorded in well differentiated cells, including normal gating mode (BK_N channels). The vast majority of BK channels expressed distinctive properties, the most conspicuous being their buzz gating mode (BK_B channels). BK_B channels were insensitive to a concentration of charybdotoxin that completely and reversibly blocked BK_N channels. In contrast with the strict dependence of BK_N channel activation on cytoplasmic Ca, BK_B channels displayed substantially high open probability (P _o) after inside-out patch excision in a Ca-free medium. Intracellular trypsin down-regulated the P _o of BK_B channels, which then exhibited a greater sensitivity to cytoplasmic Ca, mainly in the positive direction (increased P _o with increased Ca). This suggested a modulatory role for Ca as opposed to its gating role in BK_N channels. Ca ions also reduced current amplitude of both types of channels. BK_B channels were less voltage sensitive than BK_N channels, but this was not correlated with their lower Ca sensitivity. We speculate that BK_B channels may represent immature forms in the developmental expression of BK channels. Received: 1 August 1995 /Received after revision: 24 October 1995 /Accepted: 25 October 1995     

甘油二酯激酶γ参与大鼠胚胎脑神经上皮细胞的发育

目的探讨甘油二酯激酶γ(DGKγ)在大鼠胚胎脑神经上皮发育中的作用及可能机制。方法 18只胎龄(E)11.5、E12.5、E14.5、E16.5、E18.5大鼠胚胎用于Western blotting方法检测DGKγ在大鼠胚胎脑组织的表达,E9.5~E18.5大鼠胚胎各5只用于免疫组织化学和免疫荧光染色方法检测DGKγ在大鼠胚胎脑的分布以及DGKγ与Ki67、乙酰胆碱转移酶(Ch AT)及酪氨酸羟化酶(TH)的细胞内共定位。结果 DGKγ蛋白含量在E11.5~E14.5表达逐渐增多,E16.5表达较E14.5显著减少,E18.5表达显著增多且高于E14.5水平。E10.5~E12.5,DGKγ在脑泡壁出现表达,并随着脑泡的发育表达在端脑、除大脑脚外的中脑、后脑和末脑;E13.5~E14.5,DGKγ的强阳性表达从海马及周围的新皮质、苍白球、嗅脑和大脑脚阳性延伸到除新皮质外的脑的各区域;E15.5至出生前,DGKγ在新皮质阳性表达增强,但在大脑脚和脑桥表达减弱。免疫荧光染色显示,E14.5,与海马和苍白球相比,嗅脑的DGKγ阳性细胞中Ki67阳性率较高(87%),Ch AT阳性率较低(9%)(均P0.05),中脑顶盖的DGKγ阳性细胞中62%为TH阳性;E16.5,新皮质的DGKγ阳性细胞中Ki67阳性率为26%,较E14.5时(49%)显著降低(P0.05),Ch AT阳性率为70%,较E14.5时(45%)显著增高(P0.05);DGKγ多位于神经上皮细胞的胞质,也可见于胞膜上,或者同时位于胞质和胞核。结论 DGKγ在胚胎脑的表达模式和亚细胞分布,提示其参与了胚胎脑神经上皮的增殖、迁移和分化后神经元的发育。     

Immunolocalization of VEGF/VEGFR system in human fetal vomeronasal organ during early development

The vomeronasal system (VNS) is an accessory olfactory structure present in most mammals adhibited to the detection of specific chemosignals implied in social and reproductive behavior. The VNS comprises the vomeronasal organ (VNO), vomeronasal nerve and accessory olfactory bulb. VNO is characterized by a neuroepithelium constituted by bipolar neurons and supporting and stem/progenitor cells. In humans, VNO is present during fetal life and is supposed to possess chemoreceptor activity and participate in gonadotropin-releasing hormone neuronal precursor migration toward the hypothalamus. Instead, the existence and functions of VNO in postnatal life is debated. Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) have been demonstrated to play fundamental roles in various neurogenic events. However, there are no data regarding the localization and possible function of VEGF/VEGFRs in human fetal VNO. Therefore, this study was conceived to investigate the expression of VEGF/VEGFRs in human VNO in an early developmental period (9–12 weeks of gestation), when this organ appears well structured. Coronal sections of maxillofacial specimens were subjected to peroxidase-based immunohistochemistry for VEGF, VEGFR-1 and VEGFR-2. Double immunofluorescence for VEGF, VEGFR-1 or VEGFR-2 and the neuronal marker protein gene product 9.5 (PGP 9.5) was also performed. VEGF expression was evident in the entire VNO epithelium, with particularly strong reactivity in the middle layer. Strongly VEGF-immunostained cells with aspect similar to bipolar neurons and/or their presumable precursors were detected in the middle and basal layers. Cells detaching from the basal epithelial layer and detached cell groups in the surrounding lamina propria showed moderate/strong VEGF expression. The strongest VEGFR-1 and VEGFR-2 expression was detected in the apical epithelial layer. Cells with aspect similar to bipolar neurons and/or their presumable precursors located in the middle and basal layers and the detaching/detached cells displayed a VEGFR-1 and VEGFR-2 reactivity similar to that of VEGF. The basal epithelial layer exhibited stronger staining for VEGFRs than for VEGF. Cells with morphology and VEGF/VEGFR expression similar to those of the detaching/detached cells were also detected in the middle and basal VNO epithelial layers. Double immunofluorescence using anti-PGP 9.5 antibodies demonstrated that most of the VEGF/VEGFR-immunoreactive cells were neuronal cells. Collectively, our findings suggest that during early fetal development the VEGF/VEGFR system might be involved in the presumptive VNO chemoreceptor activity and neuronal precursor migration.     

Neural plate- and neural tube-forming potential of isolated epiblast areas in avian embryos

Summary Formation, shaping, and bending of the neural plate and closure of the neural groove are complex processes resulting in formation of the neural tube. Two experiments were performed using avian embryos as model systems to examine these events. First, we transected blastoderms near the level of Hensen's node to determine the potential of prenodal neural plate to form neural tube in isolation from primitive streak regression. Our results demonstrate that shaping and bending of the prenodal neural plate occur under these conditions, but neural groove closure is inhibited. Second, we isolated various areas of postnodal epiblasts to determine their potential to form neural plate. Our results suggest that the area of the postnodal epiblast that can form neural plate consists of paired tracts lying adjacent to the definitive primitive streak and extending caudally at least 1 mm from its cranial end.     

Altered mitochondrial morphology of rat embryos in diabetic pregnancy

Background: Previous studies in vivo and in vitro have suggeted that the oxidative metabolism of the embryo may have a role in the treatogenicity of diabetic pregnancy. In particular, the production of reactive oxygen species by the embroyonic mitochondiria has been implicated in the teratological process. The induction of congenital malformations by the diabetic milieu occurs during the early embryonic development. The present study aimed to estimate the role of the embryonic mitochondria in the teratological process of diabetic pregnancy by studying mitochondrial morphology in the embryos exposed to a diabetic environment in vivo or in vitro during early organogenesis and late fetal development. Methods: For studies in vivo embryos of control or streptozotocin-dia-betic rats were taken at gestational days 9–11 and sujected to light and electron microscopical analysis. The brain, heart, and liver of day-15 fetuses were also observed. For studies in vitro day-9 embryos of normal rats were cultured in a whole-embryo culture system for 48 hours. The culture media were supplied with high conectration of diabetes-related substrates and metabolites, and their effect on structure of embryonic neuropeithelial cells determined. Results: The light microscopoical observations demonstrated numberous cytoplasmic vaculoes in the ectoderm of day-9 embryos and the neuroepithelium and blood cells of day-10 and day-11 embryos of diabetic rats. Ultrastructuraily, these vacuoles were found to be mitochondria undergoing large-amplitude swelling with loss of matrix density and disturbed cristae. In contrast, no Mitochondrial differences were found in the brain, heart, and liver, when day-15 fetuses from normal and diabeic rats were compared. Ultrastructural analysis of day-9 embryos cultured for 48 hours in the presnce of high conectrations of D-Glucose, pyruvate, β-hydroxybutyrate, and α-ketoisocaproate also showed high-amplitude mitochondrial swelling in the neuroepithelium. The motochondrial swelling was, however, not found in embryos cultured in a high conectration of L-glucose, excluding simple osmotic effects of the diabetes-related substrated and metabolites. Conclusions: The mitochondrial morphological changes appeared in embryos subjected to a diabetic environment during a time period when the congenital malformations in diabetic pregnancy are induced. The results support the notion that embryonic mitochondria are involved in the teratological process of diabetic pregnancy. © 1995 Wiley-Liss, inc.     

Information provided by the skeletal muscle and associated neurons is necessary for proper brain development

Previously, motor cortex of term Myf5(-/-):MyoD(-/-) fetuses (e.g. have ablated skeletal myogenesis and consequent early loss of lower motor and proprioceptive neurons) was found to lack giant pyramidal cells. We further investigated how the absence of the extrinsic stimuli from the lacking structures influences brain development. Apparently normal motor cortex of mutant fetuses was found to have dramatically reduced presence of nestin-expressing processes of neural precursors, calretinin-expressing pyramidal neurons and calbindin-expressing neurons. Consistently, some areas of the extrapyramidal tract had significantly decreased number of differentiated neurons in mutant brains. Surprisingly, we were unable to detect any change in proliferation or cell death in the mutant neuroepithelium. Together, it appears that the information provided by the lacking structures influences the ratios of the differentiated neuronal types and their progenitor cells.     

The developing neuroepithelium in human embryonic and fetal brain studied with vimentin-immunocytochemistry

Summary The neuroepithelial cells, which constitute the primordium of the CNS, are potentially capable of generating neuronal and glial cell lineages concomitantly. The appearance and morphological development of vimentin-positive neuroepithelial cells in human embryonic and fetal brain (4–16 weeks) were studied with immunocytochemistry. In embryos aged 4–6 weeks, vimentin-reactivity was seen in all neuroepithelial cells, including those which exhibited mitotic figures. The distribution of reactivity changed according to a general developmental pattern, which commenced and proceeded temporally different in various regions of the CNS. All regions exhibited vimentin-positive neuroepithelial cells, the distribution and morphology of which gradually changed, resulting in lamination of the neural wall into two and subsequently three layers. The neocortex and midline raphe were the only regions to differ significantly from the general pattern. When reactivity to glial fibrillary acidic protein developed at 7–8 weeks, the distribution was very much like that of vimentin at the same stage. Reactivity to glial, neuronal and other cellular markers (S-100, neurofilament, neuron specific enolase, desmin, and cytokeratin) revealed different distributions. Although cells retaining vimentin beyond the ventricular zone stage are radial glial cells and presumptive fibrous astrocytes, it seems unlikely that vimentin is a marker for a distinct cell lineage during early CNS development. It is suggested that all neuroepithelial cells in vivo differentiate to a stage where they express vimentin, and that vimentin may have a functional role in cellular movements and during the interkinetic nuclear migration.     

Apical cell escape from the neuroepithelium and cell transformation during terminal lip fusion in the house shrew embryo

The house shrew embryo has many cells in the ventricular lumen and on the luminal surface of the fusing terminal lip of the cephalic neural tube. The origin and fate of these cells were studied by means of light and electron microscopy, and by DiI labeling in a whole-embryo culture system. The cells appeared at stage 11A and persisted until stage 12A. Most of the cells seemed to originate from the neuroepithelium, as shown by frequent observations of epithelial cell escape and DiI labeling analysis. The cells on the luminal surface sometimes showed apoptotic features, but were not subjected to phagocytosis. Some of the escaping cells seemed to migrate to the ventral part of the prosencephalic neuropore and insert themselves into it. Others separated from the luminal surface and floated into the lumen. It seems likely that the floating cells either become autolyzed, or else change into macrophage-like cells, the latter alternative being supported by the results of DiI labeling. The macrophage-like cells actively phagocytosed the other degenerating cells and apoptotic bodies. These observations suggest that the apical escape of cells may play an important role in the remodeling of the neural fold during the terminal lip fusion, and that early neuroepitheial cells may have the potential to become cells with vigorous phagocytic activity, like macrophages.     

Early embryonic brain development in rats requires the trophic influence of cerebrospinal fluid

Cerebrospinal fluid has shown itself to be an essential brain component during development. This is particularly evident at the earliest stages of development where a lot of research, performed mainly in chick embryos, supports the evidence that cerebrospinal fluid is involved in different mechanisms controlling brain growth and morphogenesis, by exerting a trophic effect on neuroepithelial precursor cells (NPC) involved in controlling the behaviour of these cells. Despite it being known that cerebrospinal fluid in mammals is directly involved in corticogenesis at fetal stages, the influence of cerebrospinal fluid on the activity of NPC at the earliest stages of brain development has not been demonstrated. Here, using “in vitro” organotypic cultures of rat embryo brain neuroepithelium in order to expose NPC to or deprive them of cerebrospinal fluid, we show that the neuroepithelium needs the trophic influence of cerebrospinal fluid to undergo normal rates of cell survival, replication and neurogenesis, suggesting that NPC are not self-sufficient to induce their normal activity. This data shows that cerebrospinal fluid is an essential component in chick and rat early brain development, suggesting that its influence could be constant in higher vertebrates.     

Fucosylated glycans in the periventricular structures and the cerebrospinal fluid of the fetal rat forebrain. An autoradiographic and lectin binding histiotopic study

Our autoradiographic 3H-fucose incorporation study of the brains of 20-day-old rat fetuses showed that the synthesis of fucosylated glycans is significantly higher in the ventricular germinative zone of the forebrain hemisphere than in the more superficial layers, including the cortical plate. Intense incorporation of 3H-fucose also occurred in the choroid plexus, both its epithelial and stromal component, in the primordial ependymal lining of the lateral ventricles, meninges and capillaries of the forebrain parenchyma. In the lateral ventricles, densely labeled microprecipitates of the cerebrospinal fluid (CSF) were occasionally observed. The histiotopic differences in 3H-fucose labeling were absent, or were much less expressed, in the autoradiograms prepared from unfixed cryostat sections containing mainly unincorporated isotope. This indicates that the blood-mediated supply of 3H-fucose to the studied brain compartments was essentially equal and our incorporation data reflect actual differences in the rate of fucosylation within the forebrain hemispheres. The cytochemical lectin-binding assay, carried out with Ulex europaeus and Lotus tetragonolobus agglutinins, showed that regions with a higher rate of 3H-fucose incorporation were also richer in fucose-bearing glycoconjugates. The study revealed that the periventricular regions and the CSF of fetal rat forebrain form a fucosylated glycan-enriched complex, which represents a new chemoarchitectonic feature that may be of importance for maintaining the germinative properties of the ventricular neuroepithelium and the growth of the hemispheric ventricles.