Dong-Ming Yu

Prenatal Alcohol Exposure Induces Long-Term Changes in Dendritic Spines and Synapses in the Mouse Visual Cortex

AIMS To study the long-term changes of dendritic spine and synapse taking place in a mouse model of fetal alcohol spectrum disorders (FASDs). METHODS Pregnant mice were intubated daily with ethanol (EtOH) from E5 to parturition. A DiI diolistic method was used to label dendritic spines of pyramidal cells in the visual cortex of EtOH-exposed and control pups over the period from postnatal (P) day P0 to P30; synaptic ultrastructure was also analyzed using transmission electron microscopy. RESULTS Prenatal alcohol exposure was associated with a significant decrease in the number of dendritic spines of pyramidal neurons in the visual cortex and an increase in their mean length. The changes were dose dependent and persisted to P30. Ultrastructural changes were also observed, with decreased numbers of synaptic vesicles, narrowing of the synaptic cleft and thickening of the postsynaptic density compared to controls; ultrastructural changes also persisted to P30. CONCLUSIONS Prenatal alcohol exposure is associated with long-term changes in dendritic spines and synaptic ultrastructure; these alterations probably reflect the developmental retardation of dendritic spines and synapses in visual cortex. These long-term changes are likely to contribute to lifelong mental retardation associated with childhood FASDs.

Synaptogenesis in the developing mouse visual cortex

We used transmission electron microscopy to study ultrastructural changes accompanying synaptogenesis in the fetal and postnatal mouse visual cortex (primary visual cortex). Immunostaining and DiI diolistic assay were also employed in order to evaluate synaptophysin expression and dendritic spine development. Nascent synapses were seen as early as E15, although these were immature and were composed of a presumed presynaptic terminal with pleiomorphic vesicles in the vicinity of a partner cell body or projection. The postsynaptic plasmalemma remained unspecialized and the gap between pre- and post-synaptic plasmalemmas was only 5-10nm, significantly narrower than the mature synaptic cleft. With increasing age there was gradual thickening of both the pre- and post-synaptic membranes, with widening of the synaptic cleft to 15-20 nm. Ultrastructurally mature synapses were not seen until P7; at this time both Grays type I and II could be observed. Synaptogenesis correlated with the development of synaptic spines and synaptophysin expression. Because synapse maturation was synchronous with dendritic spine differentiation, synaptic specialization may be dependent on dendritic spine maturation and the expression of presynaptic vesicle components. In the meantime, the study also indicated that the synaptogenesis was connected with the development and maturation of neocortex.

The tracing study of developing entorhino-hippocampal pathway

The entorhino‐hippocampal pathway is the major excitatory input from neurons of the entorhinal cortex on both ipsilateral and contralateral hippocampus/dentate gyrus. This fiber tract consists of the alvear path, the perforant path and a crossed commissural projection. In this study, the histogenesis and development of the various subsets of the entorhino‐hippocampal projection have been investigated. DiI, DiO, Fast Blue tracing and calretinin immunocytochemistry as well as were carried out with pre and postnatal rats at different developmental stages. The alvear path and the commissural pathway start to develop as early as embryonic day E16, while the first perforant afferents reach the stratum lacunosum‐moleculare of the hippocampus at E17 and at outer molecular layer of the denate gyrus at postnatal day 2. Retrograde tracing with DiI identifies entorhinal neurons in layer II–IV as the developmental origin of the entorhino‐hippocampal pathway. Furthermore, calretinin immunocytochemistry revealed transitory Cajal‐Retzius cells in the stratum lacunosum‐moleculare of the hippocampus from E16. DiI labeling of entorhinal cortex fibers and combined calretinin‐immunocytochemistry reveal a close relationship between Cajal‐Retzius cells and entorhinal afferents. This temporal and spatial relationship suggests that Cajal‐Retzius cell serves as a guiding cue for entorhinal afferents at early cortical development.

Neuronal Apoptosis in the Developing Cerebellum

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Expression of the apoptosis-related proteins caspase-3 and NF-κB in the hippocampus of Tg2576 mice

To investigate the relations between neuroapoptosis and the onset and development of Alzheimer’s disease (AD), especially the role of NF-κB in the regulation of neuroapoptosis. Caspase-3 and NF-κB (p50) expressions in the CA3 region of the hippocampus in APPswe Tg2576 transgenic mice were studied from postnatal day 0–180, using Nissl staining, immunohistochemistry and RT-PCR methods. Both neuronal apoptosis and NF-κB activity decreased gradually with the increase of age in wild type and Tg2576 mice. However, the number of caspase-3-positive or NF-κB-positive pyramidal cells in Tg2576 mice was greater than that in age-matched wild type mice, with significant differences after postnatal day 14 (P < 0.01 or P < 0.05). Linear regression analyses of caspase-3 and NF-κB expression demonstrated a correlation between neuroapoptosis and activity of NF-κB. The process of neuroapoptosis is consistent with the onset and development of AD. Furthermore, the observed correlation between neuroapoptosis and NF-κB activity suggests a role of NF-κB in hippocampal neuroapoptosis. 探讨阿尔茨海默病(Alzheimer’s disease, AD)的发生、 发展与神经细胞凋亡之间的规律, 尤其是NF-κB在神经细胞凋亡中的调节作用。 以0–180日龄的Tg2576转基因小鼠作为研究对象, 正常野生型小鼠为对照, 应用caspase-3 和NF-κB 免疫组织化学、 免疫荧光双标、 Nissl 染色、 RT-PCR 等方法进行研究。 结果 随着小鼠日龄的增长, 对照组与模型组小鼠的海马CA3区锥体细胞中, 凋亡细胞密度和NF-κB阳性细胞密度逐渐下降; 出生14 d 以后, 模型组凋亡细胞密度和NF-κB阳性细胞密度均高于对照组(P < 0.05), 且小鼠海马caspase-3 mRNA表达水平与caspase-3 免疫组织化学结果基本吻合。 模型组NF-κB 和caspase-3 的表达水平均高于对照组。 凋亡相关基因NF-κB和caspase-3在AD的发病机制中可能起重要作用, NF-κB与神经细胞凋亡有正相关关系。 此外, caspase-3 与NF-κB 共同表达于同一细胞中, 提示NF-κB 的激活可能参与神经细胞凋亡的调节。

Radial glial cells and the lamination of the cerebellar cortex.

Radial glial cells are stem cells that play an important role in neuronal migration and proliferation in the developing brain. However, how radial glial cells contribute to the lamination of the cerebellar cortex is not well understood. We therefore used immunohistochemistry and BrdU labeling to follow radial glial cell differentiation, cell migration and cerebellar cortex development in mice from embryonic day 8 to postnatal day 180. We report that radial glial cells represent the stem cell population of the neuroepithelium of the neural tube, and act as progenitors for both neurons and neuroglia. In addition, radial glial cells not only give rise to the principal cells of the cerebellar cortex, the Purkinje and granule cells, but they also provide a scaffold for the migration of these cells. We conclude that radial glial cells play a pivotal role in establishing the laminar structure of the cerebellar cortex.

Characterization of hippocampal Cajal-Retzius cells during development in a mouse model of Alzheimer's disease (Tg2576).

Cajal-Retzius cells are reelin-secreting neurons in the marginal zone of the neocortex and hippocampus. The aim of this study was to investigate Cajal-Retzius cells in Alzheimers disease pathology. Results revealed that the number of Cajal-Retzius cells markedly reduced with age in both wild type and in mice over-expressing the Swedish double mutant form of amyloid precursor protein 695 (transgenic (Tg) 2576 mice). Numerous reelin-positive neurons were positive for activated caspase 3 in Tg2576 mice, suggesting that Cajal-Retzius neuronal loss occurred via apoptosis in this Alzheimers disease model. Compared with wild type, the number of Cajal-Retzius cells was significantly lower in Tg2576 mice. Western blot analysis confirmed that reelin levels were markedly lower in Tg2576 mice than in wild-type mice. The decline in Cajal-Retzius cells in Tg2576 mice was found to occur concomitantly with the onset of Alzheimers disease amyloid pathology and related behavioral deficits. Overall, these data indicated that Cajal-Retzius cell loss occurred with the onset and development of Alzheimers disease.

Reelin, a guidance signal for the regeneration of the entorhino-hippocampal path.

The importance of reelin for cortical lamination in the developing CNS is well established, but its role in nerve fiber growth is not well understood. In this study, hippocampal slices were co-cultured with entorhinal slices of GFP mice, in order to compare the growth of the entorhino-hippocampal path in wild type and reeler mice. On day 6, regenerated fibers were seen to navigate from the entorhinal cortex into the hippocampus. The results showed that in wild type mice, regenerated fibers grew along the molecular layer of the dentate gyrus, and only a few fibers were found to penetrate through the granular layer into the hilus. This specific orientation was similar to the perforant path in vivo. Compared with perforant path regeneration in wild type mice, reeler mice seemed to have lost their specific orientation and proper termination in the hippocampus. Without the guidance signal from reelin, the regenerated fibers left the molecular layers and continued to grow aberrantly, i.e., in the granular layer, hilus, pyramidal layer and even stratum oriens. Particularly in the dentate gyrus, the fibers meandered around the cells in the hilus and resembled a network. The study concludes that reelin also serves as an important guidance signal for neuroregeneration of the perforant path.

Death Receptor 5 and Neuroproliferation

Tumor necrosis factor-related apoptosis-inducing ligand or Apo2 ligand is a member of the tumor necrosis factor superfamily of cytokines that induces apoptosis upon binding to its death domain-containing transmembrane receptors, death receptors 4 and 5 (DR4, DR5). However, DR5 is also expressed in the developing CNS where it appears to play a role unrelated to apoptosis, and instead may be involved in the regulation of neurogenesis. We report on the distribution of DR5 expression in mouse hippocampus, cerebellum, and rostral migratory stream (RMS) of olfactory bulb from embryonic (E) day 16 (E16) to postnatal (P) day (P180). At E16, DR5-positive cells were distributed widely in embryonic hippocampus with strong immunostaining in the developing dentate gyrus. In newborn hippocampus, DR5-positive cells were predominantly located in proliferative zones, such as dentate gyrus, subventricular zone, and RMS. After postnatal day 7 (P7), the number of DR5-positive cells decreased, and cells with intense fluorescence were primarily restricted to the subgranular layer (SGL), although the granular cell layer showed weak fluorescence. After P30, only few DR5-positive cells were found in SGL, and mature granule cells were negative for DR5 expression. To address whether DR5 expression is a restricted to progenitor cells and newborn neurons, we performed 5-bromo-deoxyuridine labeling. We report that proliferative cells in the SGL selectively express DR5, with lower levels of expression in cells positive for doublecortin, a marker of newborn neurons. In addition, the stem cells in intestine, cerebellum, and RMS were also demonstrated to be DR5-positive. In the meantime, in cerebellum, DR5-positive cells were also positive for glial fibrillary acidic protein, a marker of proliferative Bergmann cells. We conclude that DR5 is selectively expressed by neuroprogenitor cells and newborn neurons, suggesting that the DR5 death receptor is likely to play a key role in neuroproliferation and differentiation.

The effects of prenatal alcohol exposure on the developmental retina of mice.

AIMS Our aim is to investigate the effects of prenatal alcohol exposure (PAE) on the development of retinal bipolar and horizontal cells. METHODS The alterations of the retinal bipolar and horizontal cells in P7, P14 and P30 mice were observed after PAE, with immunofluorescent labeling and DiI diolistic assay. RESULTS The retinal development of filial pups was affected by PAE in a dose-dependent and long-term manner. The number of bipolar cells of alcohol groups was significantly lower than that of the control, and the dendritic receptive field of horizontal cells was also significantly smaller than those of the control groups (P < 0.01). CONCLUSION PAE was able to cause retarded development of pup retinal neural cells.