ApoE4 delays dendritic spine formation during neuron development and accelerates loss of mature spines in?vitro

The ε4 allele of the gene that encodes apolipoprotein E (APOE4) is the greatest genetic risk factor for Alzheimer''s disease (AD), while APOE2 reduces AD risk, compared to APOE3. The mechanism(s) underlying the effects of APOE on AD pathology remains unclear. In vivo, dendritic spine density is lower in APOE4-targeted replacement (APOE-TR) mice compared with APOE2- and APOE3-TR mice. To investigate whether this apoE4-induced decrease in spine density results from alterations in the formation or the loss of dendritic spines, the effects of neuron age and apoE isoform on the total number and subclasses of spines were examined in long-term wild-type neurons co-cultured with glia from APOE2-, APOE3- and APOE4-TR mice. Dendritic spine density and maturation were evaluated by immunocytochemistry via the presence of drebrin (an actin-binding protein) with GluN1 (NMDA receptor subunit) and GluA2 (AMPA receptor subunit) clusters. ApoE isoform effects were analyzed via a method previously established that identifies phases of spine formation (day-in-vitro, DIV10–18), maintenance (DIV18–21) and loss (DIV21–26). In the formation phase, apoE4 delayed total spine formation. During the maintenance phase, the density of GluN1+GluA2 spines did not change with apoE2, while the density of these spines decreased with apoE4 compared to apoE3, primarily due to the loss of GluA2 in spines. During the loss phase, total spine density was lower in neurons with apoE4 compared to apoE3. Thus, apoE4 delays total spine formation and may induce early synaptic dysfunction via impaired regulation of GluA2 in spines.     

下丘脑局部损毁对大鼠双侧催产素神经元爆发放电的影响

为探究授乳大鼠双侧下丘脑巨细胞催产素神经元同步化射乳反射爆发放电的中枢所中,我们采用双微电极细胞外记录技术,观察了选择性脑切割损毁后的大鼠双侧视上核内催们素神经元在仔鼠吸吮刺激下射乳反射爆发放电。结果显示：在腹侧这画以上横向民单侧中脑中部,不同能阻断双侧催产素神经元的同步化爆发放 单侧下丘脑中间内侧部横切则可阻断这种经爆发放电。这些结果表明;中脑中部至一丘脑中部这一脑区在双侧视上核内催产素神经元的     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

姜黄素对慢性低O2高CO2作用下大鼠脑组织Fas/FasL表达的影响

目的：观察慢性低02高C02作用下大鼠脑组织Fas/FasL表达及神经细胞超微结构变化,探讨姜黄素是否能影响慢性低O2高CO2作用下Fas/FasL表达以及脑神经细胞的超微结构变化。方法：将健康sD雄性大鼠15只,随机分为常氧对照组、慢性低02高c02组、慢性低O2高CO2姜黄素组（姜黄素采用灌胃给药,150mg/（kg·d）;应用免疫组织化学法和蛋白免疫印记法对脑神经细胞凋亡分子Fas/FasL的表达进行定性、定量测定,透射电镜下观察大脑皮质神经细胞超微结构形态学改变。结果：①免疫组织化学和蛋白免疫印记法显示,慢性低O2高CO2组中脑组织Fas/FasL的表达显著高于常氧对照组和慢性低O2高CO2姜黄素组。②脑组织的透射电镜结果显示,常氧对照组神经细胞及细胞器结构形态正常;慢性低O2高CO2组可见神经细胞和细胞器肿胀,细胞核染色质边集,出现凋亡小体;慢性低O2高CO2姜黄素组神经细胞核、细胞器、血管的形态结构基本正常。结论：①慢性低O2高CO2可上调脑组织Fas/FasL的表达,并诱导脑神经细胞呈现凋亡的超微结构变化;②姜黄素可能通过抑制Fas/FasL的表达减轻慢性低O2高CO2诱导的脑神经细胞的超微结构变化。     

电刺激大鼠外侧缰核对延髓甩尾相关细胞的活动和甩尾反射的影响

在浅麻醉大鼠上,在延髓腹内侧结构内观察到三种具有不同放电类型的细胞,即乃尾前放电骤停的撤反应细胞,甩尾前放电骤增的给反应细胞和甩尾无关的中性细胞。电刺激外侧缰核可抑制撤反应细胞的自发放电,加强给反应细胞自发放电,从而易化两类细胞的甩尾相关反应,同时易化伤害刺激引起的甩尾反射。实验结果说明,外侧缰核对节段性防御反射有易化作用,这种易化作用可能是通过延髓内撤反应和反应细胞的协同活动而实现的。     

Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

大鼠中枢神经系统衰老的形态学研究Ⅳ.大脑皮质锥体细胞树突棘的数量变化

用6、12与31个月的雄性Wistar大鼠的大脑Krieg 2、3区皮质,对其V层大锥体细胞的五段50μm长度内的树突棘做形态学定量研究。在Golgi法的切片中共计数了三个年龄组的151个细胞的725段树突的棘密度。结果表明,老年大鼠比成年和青年大鼠的棘密度普遍下降。其中以基树突与侧树突棘度下降最显著(减少24％左右),顶树突只中段有明显减少。老年大鼠锥体细胞还常出现胞体、树突及其分支的明显形态改变。     

A Cell Culture Model for Androgen Effects in Motor Neurons

Abstract: Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo. To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells. Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype. When differentiated in the presence of androgen, AR-expressing cells, but not control cells, exhibit a dose-dependent change in morphology: androgen-treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers. In addition, androgen promotes the survival of AR-expressing cells, but not control cells, under low-serum conditions. Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.     

N‐cadherin regulates the proliferation and differentiation of ventral midbrain dopaminergic progenitors

Adherens junction (AJ) between dopaminergic (DA) progenitors maintains the structure of ventricular zone and polarity of radial glia cells in the ventral midbrain (vMB) during embryonic development. However, it is unclear how loss of N‐cadherin might influence the integrity of the AJ and the process of DA neurogenesis. Here, we used conditional gene targeting approaches to perform the region‐specific removal of N‐cadherin in the neurogenic niche of DA neurons in the vMB. Removal of N‐cadherin in the vMB using Shh‐Cre disrupts the AJs of DA progenitors and radial glia processes in the vMB. Surprisingly, loss of N‐cadherin in the vMB leads to a significant expansion of DA progenitors, including those expressing Sox2, Ngn2, and Otx2. Cell cycle analyses reveal that the cell cycle exit in the progenitor cells is decreased in the mutants from E11.5 to E12.5. In addition, the efficiency of DA progenitors in differentiating into DA neurons is decreased from E10.5 to E12.5, leading to a marked reduction in the number of DA neurons at E11.5, E12.5, and E17.5. Loss of N‐cadherin leads to the diffuse distribution of β‐catenin proteins, which are a critical component of AJ and Wnt signaling, from the AJ throughout the entire cytoplasm in neuroepithelial cells, suggesting that canonical Wnt signaling might be activated in the DA progenitors in vMB. Taken together, these results support the notion that N‐cadherin regulates the proliferation of DA progenitors and the differentiation of DA neurons through canonical Wnt‐β‐catenin signaling in the vMB. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 518–529, 2013     

A Histochemical Examination of the Staining of Kainate-Induced Neuronal Degeneration by Anionic Dyes

Anionic dyes, notably acid fuchsine, strongly stain the nuclei and cytoplasm of neurons severely damaged by injury or disease. We provide detailed instructions for staining nervous tissue with toluidine blue and acid fuchsine for optimal demonstration of injured neurons. Degeneration was induced in the hippocampus of the mouse by systemic administration of kainic acid, and the resulting acidophilia was investigated using paraffin sections of the Carnoy-or Bouin-fixed brains. The affected cells were bright red with the toluidine blue-acid fuchsine sequence. Their nuclei were stainable also with alkaline Biebrich scarlet and with the 1,2-naphthoquinone-4-sulfonic acid-Ba(OH)₂ method; all staining was blocked by benzil but was relatively refractory to deamination by HNO₂. These properties indicated an arginine-rich protein. The nuclei were strongly acidophilic in the presence of a high concentration of DNA (strong Feulgen reaction), and acidophilia could not be induced in normal neuronal nuclei by chemical extraction of nucleic acids. The cytoplasmic acidophilia of degenerating hippocampal neurons was due to a protein rich in lysine (extinguished by alkalinity, easily prevented by deamination, and unaffected by benzil). Stainable RNA was absent from the perikarya of the affected cells, but normal neuronal cytoplasm did not become acidophilic after extraction of nucleic acids. We suggest that kainate-induced cell death is preceded by increased production of basic proteins, which become concentrated in the nucleus and perikaryon. Groups of small, darkly staining neurons were seen in the cerebral cortex in control and kainite-treated mice. These shrunken cells were purple with the toluidine blue-acid fuchsine stain, and were attributed to local injury incurred during removal of the unfixed brain.