利用单感器记录与神经元示踪结合对棉铃虫主要性信息素感器内神经元投射的鉴定

【目的】鉴定雄性棉铃虫Helicoverpa armigera成虫触角性信息素感器嗅觉受体神经元的功能、形态及中枢投射路径。【方法】利用单感器记录技术记录棉铃虫嗅觉受体神经元对性信息素的反应,同时采用荧光染料作为示踪剂染色标记嗅觉受体神经元;使用免疫组织化学方法处理相应的脑组织,标记脑内触角叶的神经纤维球结构;用激光扫描共聚焦显微镜获取图像数据,使用图形软件ZEN和Amira4.1.1进行三维结构重建。【结果】记录到雄性棉铃虫成虫触角上长毛形感器对主要性信息素成分Z11-16∶Ald产生明显的电生理反应,并成功染色标记了该感器内的嗅觉受体神经元。染色标记显示该感器内具有两个嗅觉受体神经元,其轴突通过触角神经分别投射触角叶内的云状体神经纤维球和普通神经纤维球。【结论】单感器记录与神经元示踪两技术结合能够用于鉴定昆虫触角嗅觉受体神经元的功能、形态和投射至神经纤维球的路径。与赖氨酸钴方法比较,使用荧光染料法进行神经元示踪,操作更简便,且易于进行三维空间分析,为调查棉铃虫其他嗅觉神经元的投射路径以明确外周气味受体感受与中枢系统的联系提供了有力技术支持。     

Factors that influence the development of cultured neurons from the brain of the mothManduca sexta

Summary During metamorphic adult development, neurons and glial cells in the developing olfactory (antennal) lobes of the moth undergo characteristic and extensive changes in shape. These changes depend on an interplay among these two cell types and ingrowing sensory axons. All of the direct cellular interactions occur against a background of changing steroid hormone titers. Antennal-lobe (AL) neurons dissociated from stage-5 (of 18 stages) metamorphosing animals survive at least 3 wk in primary cell culture. We describe here the morphological influences on AL neurons of (1) exposure to the steroid hormone 20-hydroxyecdysone, (2) exposure to sensory axons, and (3) interactions among the AL neurons. Cultured AL neurons respond only weakly, if at all, to 20-hydroxyecdysone. They do, however, show greater total outgrowth and branching when they had been exposed in vivo to sensory axons. Because there is no direct contact between some of the neuronal types and the sensory axons at the time of dissociation, the increase in outgrowth must have been mediated via a diffusible factor(s). When AL cells (neurons and glia) are plated at high density in low volumes of medium, or when the cells are plated at low density but in the presence of medium conditioned by high-density cultures, neurite outgrowth and cell survival are increased. Nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor-basic (bFGF), transforming growth factor-β (TGF _β ) and insulin-like growth factor (ILGF) had no obvious effect on neuronal morphology and thus are unlikely to underlie these effects. Our results suggest that the mature shape of AL neurons depends on developmental interactions among a number of diffusible factors.     

Low doses of a neonicotinoid insecticide modify pheromone response thresholds of central but not peripheral olfactory neurons in a pest insect

Insect pest management relies mainly on neurotoxic insecticides, including neonicotinoids, leaving residues in the environment. There is now evidence that low doses of insecticides can have positive effects on pest insects by enhancing various life traits. Because pest insects often rely on sex pheromones for reproduction, and olfactory synaptic transmission is cholinergic, neonicotinoid residues could modify chemical communication. We recently showed that treatments with different sublethal doses of clothianidin could either enhance or decrease behavioural sex pheromone responses in the male moth, Agrotis ipsilon. We investigated now effects of the behaviourally active clothianidin doses on the sensitivity of the peripheral and central olfactory system. We show with extracellular recordings that both tested clothianidin doses do not influence pheromone responses in olfactory receptor neurons. Similarly, in vivo optical imaging does not reveal any changes in glomerular response intensities to the sex pheromone after clothianidin treatments. The sensitivity of intracellularly recorded antennal lobe output neurons, however, is upregulated by a lethal dose 20 times and downregulated by a dose 10 times lower than the lethal dose 0. This correlates with the changes of behavioural responses after clothianidin treatment and suggests the antennal lobe as neural substrate involved in clothianidin-induced behavioural changes.     

Encoding of plant odour information in insects: peripheral and central mechanisms

Insects are suitable model organisms for studying mechanisms underlying olfactory coding and olfactory learning, by their unique adaptation to host plants in which the chemical senses are essential. Recent molecular biological studies have shown that a large number of genes in insects and other organisms are coding for olfactory receptor proteins. In general, one receptor type seems to be expressed in each neurone. The functional characterisations of olfactory receptor neurones have been extensive in certain insect species, demonstrating a fine-tuning of single neurones to biologically relevant odourants; both insect and plant produced volatiles. Stained neurones of the same functional type have been shown to project in one and the same glomerular unit in the primary olfactory centre, the antennal lobe. This corresponds to molecular biological studies, showing projections in one glomerulus by neurones expressing the same receptor type. Comparison of these findings with physiological and morphological characterisations of antennal lobe neurones has indicated correspondence between input and output of the glomerular units. Examples are presented from studies of heliothine moths. From the antennal lobe, the olfactory information is further conveyed to the mushroom bodies, particularly important for learning, and the lateral protocerebrum, a premotoric area. The three brain areas are regions of synaptic plasticity important in learning of odours, which is well studied in the honeybee but also in species of moths.     

Increased behavioral and neuronal sensitivity to sex pheromone after brief odor experience in a moth

Plasticity in the response to stimuli related to food and oviposition cues is well documented in insects. However, responses to cues related to reproduction, for example, sex pheromones, are considered to be innate and thus not affected by experience. Here we show that brief preexposure to sex pheromones, without ensuing reward, lowers the threshold for behavioral response and augments the sensitivity in antennal lobe interneurons to pheromone compared with naive male moths. Thus, the sex pheromone system in insects can be modulated by experience. In addition, we show that the behavioral attraction to sex pheromone increases after preexposure in a time-dependent manner: a short-term effect, possibly a form of sensitization, and a long-term effect after more than 24 h. The behavioral long-term effect is paralleled by an increase in sensitivity of interneurons in the primary olfactory center, whereas the peripheral olfactory system does not change its sensitivity. We hypothesize that short-term sensitization to sex pheromone serves as a kind of alert system, whereas the long-term effect improves male performance when reproductively active females are present.     

中华蜜蜂嗅叶胚后发育过程中的神经胶质

本研究通过形态解剖、免疫组织化学等技术,对中华蜜蜂Apis cerana cerana Fabricius工蜂嗅叶胚后发育中神经胶质的模式进行了比较研究。结果表明:神经胶质在蜜蜂嗅叶的发育过程中起着重要的作用,它们在嗅叶发育早期就划定了神经纤维网的边界;在嗅觉神经轴突进入嗅叶之前,对神经轴突进行"分选",并引导它们进入嗅叶特定的区域,形成神经纤维球;它们不仅规定了神经纤维网的边界和区域,还为神经纤维网提供内部的分隔。中华蜜蜂神经胶质增殖的高峰期集中在幼虫发育末期和预蛹期。     

A Conserved Role for L1 as a Transmembrane Link Between Neuronal Adhesion and Membrane Cytoskeleton Assembly

The L1-family of cell adhesion molecules is involved in many important aspects of nervous system development. Mutations in the human L1-CAM gene cause a complicated array of neurological phenotypes; however, the molecular basis of these effects cannot be explained by a simple loss of adhesive function. Human L1-CAM and its Drosophila homolog neuroglian are rather divergent in sequence, with the highest degree of amino acid sequence conservation between segments of their cytoplasmic domains. In an attempt to elucidate the fundamental functions shared between these distantly related members of the L1-family, we demonstrate here that the extracellular domains of mammalian L1-CAMs and Drosophila neuroglian are both able to induce the aggregation of transfected Drosophila S2 cells in vitro. To a limited degree they even interact with each other in cell adhesion and neurite outgrowth assays. The cytoplasmic domains of human L1-CAM and neuroglian are both able to interact with the Drosophila homolog of the cytoskeletal linker protein ankyrin. Moreover the recruitment of ankyrin to cell-cell contacts is completely dependent on L1-mediated cell adhesion. These findings support a model of L1 function in which the phenotypes of human L1-CAM mutations result from a disruption of the link between the extracellular environment and the neuronal cytoskeleton.     

昆虫嗅觉神经的计算机三维重建

基于激光扫描共聚焦显微镜平台的计算机三维重建在昆虫嗅觉神经研究中发挥了重要作用。对经荧光标记的神经组织采集系列光学切片并进行三维重建,在双翅目、鳞翅目、膜翅目、蜚蠊目昆虫中均有进展。触角叶是昆虫的初级嗅觉中心,触角叶的解剖学图谱是识别不同种和雌雄虫间嗅球体特定功能的先决条件。了解构成嗅觉传输途径的主要神经元的形态和空间关系是理解气味信息在中枢神经系统编码的基础。三维重建昆虫的嗅觉神经,对于探讨昆虫嗅觉在其寄主选择、觅食以及寻找配偶等行为中的作用具有非常重要的意义。     

The olfactory circuit of the fruit fly Drosophila melanogaster

The olfactory circuit of the fruit fly Drosophila melanogaster has emerged in recent years as an excellent paradigm for studying the principles and mechanisms of information processing in neuronal circuits. We discuss here the organizational principles of the olfactory circuit that make it an attractive model for experimental manipulations, the lessons that have been learned, and future challenges.     

昆虫嗅觉系统结构与功能研究进展

昆虫的脑由前脑、中脑和后脑组成,其中前脑含有高级感觉中枢,如蘑菇体和中央复合体,控制昆虫的学习、记忆和运动等高级神经活动;中脑包含触角叶,是嗅觉神经中心;而后脑则通常不发达,主要包括内分泌神经元和控制进食与消化的运动神经元。不同于其他物种,昆虫由于其特殊的生活习性,听觉和视觉系统相对退化,主要依赖嗅觉来捕食、交流和求偶,因此嗅觉系统尤其发达。本文综述了目前对昆虫的脑部主要神经结构和功能(中央复合体、蕈形体和触角叶结构)以及昆虫脑部结构遗传变异(性别异构,不同发育时期、不同昆虫以及昆虫与其他动物的脑部结构差异)的研究进展,并总结了目前昆虫脑对信号的加工处理和识别机制的研究结果。     

Spatial Receptive Fields for Odor Localization

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用激光共聚焦扫描技术研究昆虫触角叶的雌雄异构性

【目的】本研究探讨用激光共聚焦扫描显微镜对昆虫触角叶内结构的扫描技术。【方法】选取鳞翅目斜纹夜蛾Spodoptera litura, 蜚蠊目美洲大蠊Periplaneta americana和鞘翅目松墨天牛Monochamus alternatus, 仔细解剖得到昆虫完整脑组织, 经过Lucifer yellow染色、戊二醛固定、梯度酒精脱水和透明等一系列处理后, 用激光共聚焦扫描显微镜对昆虫触角叶结构进行分层扫描。【结果】结果显示: 经该方法处理后在激发光488 nm下能清晰扫描出昆虫触角内典型结构神经纤维球, 并且可清晰看到这3种昆虫雄性触角叶结构内的扩大型神经纤维球复合体(macroglomerular complex, MGC), 而在相应雌性昆虫体内都没有此复合体。另外通过5 μm分层扫描得到斜纹夜蛾、美洲大蠊和松墨天牛的触角叶平均厚度分别为130, 235和115 μm, 神经纤维球数量分别为35, 59和39个。【结论】激光共聚焦扫描技术是获得昆虫触角叶内部结构的一个可行方法。     

Disruption of GABAA in the insect antennal lobe generally increases odor detection and discrimination thresholds

Studies of olfactory function show that disruption of GABA A receptors within the insect antennal lobe (AL) disrupts discrimination of closely related odors, suggesting that local processing within the AL specifically enhances fine odor discrimination. It remains unclear, however, how extensively AL function has been disrupted in these circumstances. Here we psychophysically characterize the effect of GABA A blockade in the AL of the moth Manduca sexta. We used 2 GABA A antagonists and 3 Pavlovian-based behavioral assays of olfactory function. In all cases, we used matched saline-injected controls in a blind study. Using a stimulus generalization assay, we found that GABA A disruption abolished the differential response to related odors, suggesting that local processing mediates fine odor discrimination. We then assessed the effect of GABA A antagonist on discrimination thresholds. Moths were differentially conditioned to respond to one odor (reinforced conditioned stimulus [CS+]) but not a second (unreinforced conditioning stimulus [CS-]) then tested for a significant differential conditioned response between them across a series of increasing concentrations. Here, GABA A blockade disrupted discrimination of both similar and dissimilar odor pairs as indicated by generally increased discrimination thresholds. Finally, using a detection threshold assay, we established that GABA A blockade also increases detection thresholds. Because detection is a prerequisite of discrimination, this later finding suggests that disrupted discrimination may be due to impairment of the ability to detect. We conclude that the loss of ability to detect and subsequently discriminate is attributable to a loss of ability of the AL to provide a clear neural signal from background.