果蝇幼虫气味编码的分子基础

全球农业经济的损失主要来自昆虫幼虫的取食,而昆虫幼虫的取食主要是依靠气味介导的嗅觉作用。耶鲁大学的昆虫嗅觉神经生物学家对黑腹果蝇Drosophila melanogaster幼虫嗅觉分子基础进行了研究。RT—PCR扩增出23个气味受体基因,其中13个基因,同成虫触角和下颚须内的气味受体基因相同,     

甲醚菊酯和溴氰菊酯在果蝇幼虫外周神经系统的协同毒理作用

本文运用细胞内微电极记录技术研究了甲醚菊酯和溴氰菊酯对果蝇Drosophila melanogaster幼虫神经一肌肉突触兴奋性接点电位(EJP₅)的影响。用甲醚菊酯(1.49x10^-8m01/L)处理后引起果蝇EJP₅的自发释放增加和刺激后的重复后自发释放。而用溴氰菊酯(1.0x10^-8mol/L)处理的则无明显影响。这显示甲醚菊酯对果蝇外周神经主要为I型毒理作用。而溴氰菊酯则主要为Ⅱ型作用,甲醚菊酯和溴氰菊酯联合应用后,则产生兼具I型和II型特征的自发释放或诱发EJP₅发放。自发释放或重复后自发释放的频率和幅值随联合处理中甲醚菊脂和溴氰菊酯的配比而变化。这些结果说明甲醚菊酯和溴氰菊酯对果蝇幼虫外周神经的毒理具有协同作用。     

果蝇脑对2个视觉特征的清晰记忆痕迹

果蝇能够辩认和记住视觉目标。它能根据一小部分的模式参数,例如大小、颜色或者是等高方位,分析它可视环境中所选定的部分,并且贮存这些特定的参数值。像人类一样,果蝇在获得图案的过程中能够独立地识别存在于视网膜上的图案。在这里我们显示了果蝇脑一这个扇形体中心的大部分区域,包括介导视觉模式识别的网状部分。我们已经证明短期记忆有2个模式参数,即在全景中的海拔和等高方位。     

机械分离的果蝇幼虫中枢神经元全细胞钾电流的特性

培养的果蝇胚胎及幼虫中枢神经元已被广泛用于细胞膜离子通道,突触传递和胞内信使调节等电生理学研究,在本实验中,利用机械震荡分离方法获得了大量的果蝇幼虫中枢神经元,其中大部分为Ⅱ型神经元,运用膜片钳技术,鉴定了Ⅱ型神经元上五种具有不同动力学特性的全细胞钾电流,其中E型电流表型表现出与其它四种电流完全不同的“钟形”激活特性,进一步的研究还表明该类型电流具有明显的钙依赖性,而且它具有与其它四种电流不同的衰减特性。     

培养果蝇三龄幼虫的小技巧

笔者在进行果蝇唾腺染色体实验培养三龄幼虫时,根据多年的经验发现了2项很实用的小技巧.现介绍如下: 1 选择果蝇的小技巧 果蝇的选择,以野生为宜,一般不要选用长期在实验室培养的果蝇.野生果蝇通常出现在有不新鲜或腐烂的水果周围,所以水果摊或有坏烂水果的垃圾堆是捕获果蝇的首选地点,当然也可以用切成碎片的果肉招引果蝇.     

视觉和嗅觉信号对果蝇食物搜寻行为的协同作用

为了探索视觉和嗅觉信号在昆虫食物搜寻过程中的作用, 本研究利用杨梅和橘子为引诱物, 在实验室条件下测定了嗅觉和视觉信号诱集到的黑腹果蝇Drosophila melanogaster数量, 分析了嗅觉经历对果蝇嗅觉和视觉食物搜寻的影响。发现同源性嗅觉和视觉信号存在的杨梅诱集到的果蝇数量显著大于单一的视觉信号和嗅觉信号, 但异源性嗅觉和视觉信号组合诱集到的果蝇数量和单独的嗅觉信号相似。嗅觉信号预处理不仅能够显著增加嗅觉信号诱集到的果蝇数量, 其中杨梅嗅觉信号对杨梅预处理果蝇的吸引能力与视觉和嗅觉信号存在的杨梅相似, 而且异源性嗅觉和视觉信号组合诱集到的预处理果蝇数量也不低于视觉和嗅觉信号存在的杨梅。另外杨梅嗅觉信号预处理也能够显著增强杨梅视觉信号诱集到的果蝇数量。但嗅觉预处理并不会改变同源性视觉和嗅觉信号组合诱集到的果蝇数量。本研究表明, 果蝇同时利用视觉和嗅觉信号进行食物搜寻, 因此同源性视觉和嗅觉信号在果蝇诱集过程中具有协同作用。另外果蝇具有较强的记忆和学习能力, 能够将记忆中的嗅觉信号应用于食物搜寻。本研究结果不仅有利于我们了解果蝇在自然状态下的食物搜寻机制, 而且有利于开发更有效的果蝇新型诱捕器。     

果蝇视觉学习记忆能力与脑部cAMP水平的相关性

利用闭环飞行模拟系统研究果蝇视觉飞行定向行为的操作式条件化 ,证明正常果蝇视觉学习记忆能力与日龄有关 ,即 3～ 4d龄果蝇的学习记忆能力明显优于1～ 2d龄果蝇 ,蝇脑内的cAMP含量也呈现随果蝇日龄增加而增加的趋势 .同时对学习记忆缺陷型果蝇进行检测 ,其脑内cAMP含量高于正常对照组果蝇 .通过喂食PDEase抑制剂咖啡因扰乱cAMP代谢 ,使果蝇cAMP水平异常提高 ,导致果蝇学习记忆能力显著下降 ,表明果蝇视觉学习记忆需要脑内cAMP水平处于一适当范围 ,过高或过低的cAMP水平都将影响果蝇的视觉学习记忆能力     

果蝇唾液腺染色体试材准备及观察方法的改进

果蝇唾液腺观察实验中大量活体试材的准备与学生剖取腺体的操作是该实验的难点,用甲醇(或乙醇):醋酸=3:1的固定液将果蝇幼虫杀死,变活体解剖为固定后解剖,降低了实验操作的难度;将固定的幼虫置30%的甘油中,放入-20℃冰箱保存,有效地解决了大量优质试材的供给问题,提高了实验教学的效果。     

甲醛对果蝇发育和繁殖的影响

甲醛已被广泛用于人们的日常生活中,成为室内环境的主要污染源.为探讨甲醛对动物不同发育阶段的毒性,本文以模式生物果蝇Drosophila melanogaster为对象,研究了饲料中添加不同浓度甲醛对果蝇生长发育的影响.结果显示,当对照组发育至3龄幼虫（孵化后3d）时,与未添加甲醛的对照组相比,饲料中添加0.25％或0....     

Calcium/Calmodulin-Dependent Kinase II Phosphorylates Drosophila Visual Arrestin

Abstract: Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca²⁺ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. We are interested in identifying the protein kinase responsible for the phosphorylation of phosrestin I to link the transmembrane signaling to the light-adaptive response. Type II Ca²⁺/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. We show here that partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca²⁺/calmodulin-dependent kinase II and that the kinase activity exhibits regulatory properties that are unique to Ca²⁺/calmodulin-dependent kinase II such as Ca²⁺ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca²⁺/calmodulin-dependent kinase II autoinhibitory domain. We also show that Ca²⁺/calmodulin-dependent kinase II activity is present in Drosophila eye preparations. These results are consistent with our hypothesis that Ca²⁺/calmodulin-dependent kinase II phosphorylates phosrestin I. We suggest that Ca²⁺/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation.     

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage. HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.     

The optic lobe of Drosophila melanogaster

Summary We present a quantitative evaluation of Golgiimpregnated columnar neurons in the optic lobe of wildtype Drosophila melanogaster. This analysis reveals the overall connectivity pattern between the 10 neuropil layers of the medulla and demonstrates the existence of at least three major visual pathways. Pathway 1 connects medulla layer M10 to the lobula plate. Input layers of this pathway are M1 and M5. Pathway 2 connects M9 to shallow layers of the lobula, which in turn are tightly linked to the lobula plate. This pathway gets major input via M2. Pathways 1 and 2 receive input from retinula cells R1-6, either via the lamina monopolar cell L1 (terminating in M1 and M5) or via L2 and T1 (terminating in M2). Neurons of these pathways typically have small dendritic fields. We discuss evidence that pathways 1 and 2 may play a major role in motion detection. Pathway 3 connects M8 to deep layers of the lobula. In M8 information converges that is derived either from M3 (pathway 3a) or from M4 and M6 (pathway 3b), layers that get their major input from L3 and R8 or L4 and R7, respectively. Some neurons of pathway 3 have large dendritic fields. We suggest that they may be involved in the computation of form and colour. Possible analogies to the organization of pathways in the visual system of vertebrates are discussed.During the final editing of this work our friend A.P.M. Dittrich was tragically killed in an accident. Without him this and the previous work would never have been completed     

The atlas of cytoophidia in Drosophila larvae

In 2010, cytidine 50-triphosphate synthase(CTPS) was reported to form the filamentous or serpentine structure in Drosophila, which we termed the cytoophidium. In the last decade, CTPS filaments/cytoophidia have been found in bacteria, budding yeast, human cells, mice, fission yeast, plants, and archaea,indicating that this mechanism is highly conserved in evolution. In addition to CTPS, other metabolic enzymes have been identified to have the characteristics of forming cytoophidia or similar advanced structures, demonstrating that this is a basic strategy of cells. Nevertheless, our understanding of the physiological function of the cytoophidium remains incomplete and elusive. Here, we took the larva of Drosophila melanogaster as a model to systematically describe the localization and distribution of cytoophidia in different tissues during larval development. We found that the distribution pattern of CTPS cytoophidia is dynamic and heterogenic in larval tissues. Our study provides a road map for further understanding of the function and regulatory mechanism of cytoophidia.     

GAL4/UAS系统在果蝇Tis11基因RNA干扰中的应用

TTP在哺乳动物许多关键基因表达的转录后水平上起调控作用,Tis11是TTP蛋白在果蝇中的同源物.目前还没有现成的可用于研究Tis11功能的基因敲除或敲低的果蝇.为了获得肌动蛋白启动子或者热激蛋白启动子驱动表达Tis11 mRNA干扰序列的具有较高干扰效率的Tis11基因干扰果蝇,将肌动蛋白启动子或者热激启动子驱动表达的GAL4果蝇品系与融合有Tis11 mRNA干扰序列的UAS品系杂交,收集同时带有GAL4基因和UAS序列的子一代果蝇.提取所收集果蝇的总RNA,将其中的mRNA逆转录成cDNA,并设计检测Tis11基因的特异性引物,然后通过Real-time PCR检测Tis11 mRNA的表达情况.结果显示所收集的能表达Tis11基因干扰序列的子一代果蝇与不能表达Tis11基因干扰序列的对照果蝇相比,其体内Tis11 mRNA的表达水平下降明显.收集的果蝇其体内所表达的干扰序列对Tis11 mRNA干扰效果显著,我们成功获得了Tis11基因的RNA干扰果蝇.     

Visual control of straight flight in Drosophila melanogaster

Summary The optomotor system of Drosophila is investigated in a flight simulator in which the fly's yaw torque controls the angular velocity of the panorama (striped drum, negative feedback). Flies in the flight simulator maintain a stable orientation even in a homogeneously textured panorama without landmarks. During straight flight, torque is not zero. It consists of small pulses mostly alternating in polarity. The course is controlled by the duration (and possibly amplitude) of the pulses. The system operates under reafference control. By comparing the pulses with the visual input the system continuously measures and adjusts the efficacy of the torque output. The comparison, however, is not between angular velocity and yaw torque but, instead, between visual acceleration and pretorque, the first time derivative of torque. For comparison, the system first computes a cross-correlation. If the correlation coefficient is above a certain threshold the system calculates the external gain and adjusts its internal gain so as to keep the total gain constant. With the correlation coefficient below threshold, however, the system keeps the internal gain low despite the infinitely small external gain. We propose that for a reafferent optomotor system the coupling coefficient and the correlation coefficient of pretorque and visual acceleration are more relevant than the distinction between exafference and reafference.Abbreviation EMD elementary movement detector     

Neurotransmitter Receptor Ligand Binding and Enzyme Regional Distribution in the Pigeon Visual System

The relative importance of acetylcholine, dopamine, endogenous opiates, gamma-aminobutyric acid (GABA), glutamate, glycine, noradrenaline, and serotonin as transmitters in the pigeon visual system was estimated by measuring the activity of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and aromatic amino acid decarboxylase (AAD) as well as the binding of dihydroalprenolol, etorphine, kainic acid, muscimol, serotonin, spiroperidol, strychnine, and quinuclidinyl benzilate (QNB) in the tectum opticum, nucleus rotundus, ectostriatum, dorsolateral thalamus, and hyperstriatum (Wulst). As a nonvisual reference structure, the paleostriatal complex was included in the examination. The regional distribution of most of these parameters was very similar to data reported in the mammalian CNS supporting the hypothesis that the avian tectofugal and thalamofugal visual systems are homologous to the mammalian tecto-thalamo-cortical and retino-geniculo-striate pathways, respectively. On the basis of the low values of their parameters, some transmitters can be excluded as significant contributors in a number of structures. As a hypothesis for further investigations, the presence of cholinergic and serotoninergic systems in the Wulst, possibly originating in the dorsolateral thalamus and nucleus raphe, respectively, and of glycinergic and dopaminergic terminals in the paleostriatal complex is proposed.     

The Expression Patterns of Nogo-A and NgR in the Neonatal Rat Visual Nervous System

Previous studies have shown that Nogo-NgR system plays multiple roles in controlling axonal regeneration and neuronal plasticity. However, the investigation on the distributions of Nogo and NgR was limited to restricted areas or special ages, and their expression patterns in the neonatal visual nervous system is still unknown. Frozen sections were made from eyeballs and brains of three neonatal rats, and expression of Nogo and NgR proteins were examined by immunofluorescence under a laser confocal microscopy. Nogo and NgR were observed to express in the cell bodies of retina, lateral geniculate bodies and visual cortex of the neonatal rats. Our results provide evidences that the Nogo-NgR system may play an active role in the plasticity of neonatal visual system. Y. Xiaolei, Y. Rongdi and J. Shuxing contributed equally to this work.     

Axonal Transport of Glycoconjugates in the Rat Visual System

Long-Evans rats at 45 days of age were injected intraocularly with 25 mu Ci of [3H]glucosamine. Incorporation of radioactivity into retinal gangliosides, glycoproteins, and glycosaminoglycans (GAGs) was determined at various times after injection. Portions of all three classes of radioactive macromolecules were committed to rapid axonal transport in the retinal ganglion cells. With respect to gangliosides about 60% of those synthesized in the retina were retained in that structure, 30% were committed to transport to regions containing the nerve terminal structures (lateral geniculate body and superior colliculus), and about 10% were deposited in stationary structures of the axons (optic nerve and tract). With the exception of ganglioside GD3 the molecular species distribution of gangliosides synthesized in the retina matched that committed to transport. In contrast to gangliosides a smaller fraction of newly synthesized retinal glycoprotein (less than 12% of that synthesized in the retina) was committed to rapid transport to nerve ending regions and only about 0.5% was retained in the nerve and tract. The molecular-weight distribution of glycoproteins committed to transport differed quantitatively from that of the retina. With respect to GAGs an even smaller portion (1-2%) of that synthesized in the retina was committed to rapid transport; of this portion almost all was recovered in nerve terminal-containing structures. A constant proportion of each retinal GAG species was transported to the superior colliculus. We suggest that most of the retinal gangliosides are synthesized in neurons and preferentially in ganglion cells (possibly a function of the large surface membrane area supported by these cells). Subcellular fractionation experiments indicated that transported gangliosides, glycoproteins, and GAGs may be preferentially distributed into different subcellular compartments.     

利用果蝇模型研究人类心脏早期发育的分子机理(英文)

近年来 ,果蝇心脏特化的遗传机制已初步研究清楚 ,但控制人类心脏早期发育的基因尚待鉴定。因为调控果蝇和脊椎动物早期心脏细胞命运定型的途径具有保守性 ,果蝇是一种探讨人类心脏早期发育的分子机理的理想动物模式。为此目的 ,我们采用P转座子和EMS诱变技术建立了约 3 0 0 0个隐性致死基因平衡系。通过心脏前体细胞特异性抗体免疫组化筛选 ,我们检出 2 0 0余个表现心脏突变表型的平衡致死系。我们进一步利用RNAi技术对一些基因的功能进行了初步的研究 ,证明这些基因表现RNAi的突变表型 ,该类突变表型与基因突变时表现的表型相似 ,即心管呈缺陷型或无心脏前体细胞形成。利用果蝇和人类基因组计划获得的成果 ,我们从果蝇心脏侯选基因中初步克隆和鉴定了 5 0个人类同源基因 ,其中 2 0个是新基因。Northen印迹分析表明 ,一部分人类基因在心脏组织中有表达 ,从而为研究这些基因在人类心脏早期发育中的作用提供了信息。目前 ,我们正在建立转基因果蝇 ,以此为模型研究这些基因是否对心肌细胞发生或心肌功能起调控作用。产生心肌细胞突变类型的基因如果类似于人类心脏病综合症 ,则可以作为人类心脏疾病侯选基因作进一步的分析。