用“增强子陷阱”技术构造并筛选果蝇UAS/GAL4系统中GAL4新品系及脑基因表达图谱数据库的开发

"增强子陷阱"技术是建立果蝇脑全基因组表达图谱及其数据库的重要方法.筛选获得新特异表达的GAL4品系,可为进一步研究果蝇脑神经在学习记忆功能提供强有力的基因工具.通过"增强子陷阱"技术来获得果蝇突变体,并与报告转基因果蝇(UAS-EGFP)杂交,用荧光显微镜观察成年果蝇脑内荧光分布,从而获得该突变体的脑基因表达图谱,在此基础上利用JavaScript来建立果蝇脑全基因组表达数据库.目前获得基因突变体果蝇2 677种,大部分在果蝇脑中有表达,其中在果蝇嗅觉学习记忆相关脑区蘑菇体表达的基因有368个,且有部分基因特异地表达在某些传导通路上.这些果蝇基因突变体库及其表达图谱为进一步研究各基因的功能及作为遗传工具来研究各脑区结构和功能提供极大方便.     

蘑菇体-昆虫嗅觉学习和记忆中心

蘑菇体(革形体)是昆虫脑内非常重要的一个结构,构成蘑菇体的冠和叶在不同目昆虫中高度分化,其结构虽然保守,但形态上的变化在一定程度上反映了昆虫的进化地位.冠是触角叶嗅觉投射神经元的主要投射区,叶通过输出神经元联系蘑菇体与其它脑区.冠和叶在嗅觉记忆中不可或缺,垂直叶(α叶)支持长时记忆,中叶(γ叶)支持短时记忆.蘑菇体对嗅觉记忆的形成尤其是记忆的再现(提取)具有重要作用.乙酰胆碱(Ach)、γ-氨基丁酸(GABA)和一氧化氮(NO)等是蘑菇体嗅觉突触传递的主要神经递质.蘑菇体内的第二信使系统cAMP-PKA途径和NI-cGMP途径在嗅觉学习和记忆中起基础性作用.     

利用DNRX-Gal4研究果蝇Neurexin的时间和空间表达模式

果蝇Neurexin(DNRX)在突触的结构发育和突触功能上发挥着重要的作用.然而迄今为止,DNRX的时间和空间表达模式还没有被系统地研究.本研究建立了一株新的DNRX-Gal4转基因果蝇品系,评价了这株转基因Gal4品系在三龄幼虫脑中的表达模式,发现其与内源性的DNRX的表达模式是一致的.接下来利用DNRX-Gal4/UAS-Reporter系统地分析了DNRX在时间和空间上的表达模式.结果显示,DNRX在胚胎、幼虫和成虫时期均表达在中枢神经元和运动神经元,而在神经胶质细胞没有表达.在果蝇神经肌肉接头(NMJs)中,DNRX既表达在突触前也表达在突触后区域.DNRX也被发现表达在唾液腺、肠、翅膀和腿.在成虫脑中,DNRX表达在许多不同的脑区,包括蘑菇体(MBs)、触角叶(AL)和视盘.有趣的是,DNRX在控制节律的颜料释散因子(PDF)阳性的clock神经元里也有表达,同时发现DNRX在蘑菇体的表达是与果蝇的嗅觉联想式学习记忆相关的.     

昆虫记忆的形成机制及生态适应性

介绍近十几年来利用蜜蜂Apis mellifera L.、果蝇Drosophila melanogaster Meigen和寄生蜂等昆虫对学习和记忆行为研究的成果。这些研究表明昆虫的记忆形成是多阶段的。从短时记忆向长时记忆的形成过程中,cAMP反应元件结合蛋白(cAMP response element-bindingprotein,CREB)起重要的作用;而蘑菇体是学习、记忆形成的主要位点。已有的研究还表明昆虫记忆的动态是适应于不同物种的生态学需要。这些研究为探索昆虫和其它动物记忆的形成和生态适应性提供了理论基础。     

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     

果蝇在抉择过程中的行为修饰

在视觉操作条件化中, 果蝇具有惟一的行为输出——偏转扭矩, 扭矩输出参与了条件化的形成. 通过发展新的分析方法, 分析了果蝇在条件化过程中的扭矩分布模式. 果蝇扭矩分布模式的修饰过程反映了操作条件化对果蝇行为模式的影响. 先前的研究表明, 在果蝇的视觉操作条件化中, 面对矛盾的视觉线索时, 果蝇将依据不同线索的相对权重作出类似抉择的行为; 研究还表明, 果蝇脑内的蘑菇体结构在这种抉择中起重要作用. 通过对果蝇在这种类抉择行为中的扭矩输出进行“扭矩-位置分布”分析, 研究了在果蝇抉择过程中的行为模式的修饰过程. 利用这种扭矩分析方法, 得以从揭示果蝇行为模式的角度对果蝇操作式视觉联想式学习及抉择行为进行研究. 在对果蝇操作条件化中的视觉联想式学习过程、抉择过程以及行为模式修饰过程进行分析的同时, 也讨论了它们所涉及的可能不同的神经基础.     

与温度喜好行为有关的神经元

果蝇脑中蘑菇体结构内的神经元,是学习、记忆和睡眠调控等过程所必需的。Sung-Tae Hong等人所做的一项研究表明,它们在温度喜好行为中也起一定作用。一些动物,同我们人类一样,通过改变它们的代谢来从内部调控体温。但对像果蝇等其他动物来说,体温是与它们环境所进行的热交换的结果。果蝇能够本能地寻找一个与它们在遗传上所喜好的体温相匹配的环境,这个过程与哺乳动物设定固定体温的过程相似。当蘑菇体神经元中依赖于AMP的环状激酶活性被人为调低时,果蝇便不能找到它们所期望的温度;当这个活性增强时,它们便喜欢较高的温度。这项研究表明,温度感觉可能与学习和基因共享一些细胞机制。     

模拟昆虫视觉-行为抉择的强化学习模型

视觉信息用于行为抉择的过程是一个极其复杂的脑信息处理过程,昆虫或动物对外界环境的学习是以价值来控制的,并可影响其行为抉择,研究这一过程对揭示人类自身脑运行机制有重要意义.文章在郭爱克研究小组果蝇实验提供的生物依据基础上,提出了一种模拟果蝇视觉-行为抉择的神经网络模型.该模型引入了价值和基于价值的强化学习算法,应用于输入视觉图像的强化学习,以此建立果蝇脑内多巴胺和蘑菇体对于抉择判断的价值体系.模拟的结果表明,该模型可以模拟果蝇视觉信息的学习和行为抉择过程,其结果与生物实验相符,同时也为机器人视觉信息控制行为抉择的应用提供了基础.     

果蝇长时程记忆缺陷型突变体的鉴定

长时程记忆作为依赖蛋白合成的记忆组分,对于了解高等认知活动的分子机制有着重要意义.与此同时,细胞粘连分子作为影响突触可塑性的重要因子在学习与记忆研究领域也日益得到重视.为探索作用于长时程记忆的细胞粘连分子,利用P因子在果蝇基因组随机插入制造突变体,并通过大规模行为筛选得到了一个可能的长时程记忆突变体RUO. 测序结果表明,突变体RUO的P因子位于果蝇中selectin超家族对应的furrowed同源基因功能片段和未知功能的CG1806基因编码片段之间,且更靠近furrowed片段.RT-PCR结果和互补遗传学实验均表明,突变体RUO主要影响furrowed基因的表达.为了进一步确认furrowed基因与长时程记忆的相关性,引入已知的furrowed基因突变体fw1.结果表明,fw1同样具有长时程记忆缺陷,同时具备正常的学习能力.荧光共聚焦扫描成像显示,该基因特异性的表达在果蝇大脑两个对称的未知神经元中.此项工作不仅证明了furrowed基因在果蝇长时程记忆中的重要作用,而且在解剖学上揭示了果蝇神经系统中可能参与长时程记忆形成的新的神经元.     

Sciene杂志发表我科学家又一最新研究成果

2007年6月29日,国际权威学术期刊Science以报告形式发表了中科院上海生科院神经科学研究所郭爱克院士领导的学习与记忆研究组,关于多巴胺和蘑菇体环路调控果蝇基于价值的抉择的最新研究成果。这是该研究组自2001年以来,第三次在Science上发表研究论文。     

Memory impairment induced by cholinergic antagonists injected into the mushroom bodies of the honeybee

The role of honeybee central brain structures, suspected to be cholinergic, has been studied in learning and memory. The nicotinic antagonist mecamylamine and the muscarinic antagonist scopolamine were locally injected into the calyces and the alpha-lobes of mushroom bodies, and their effects on memory acquisition and retrieval were investigated using one-trial olfactory conditioning of the proboscis extension reflex. A strong impairment of the olfactory learning was noticed following mecamylamine injection into the mushroom body calyces. Mecamylamine and scopolamine disturbed retrieval processes when injected into the alpha-lobes of mushroom bodies but remain without effect on these processes when injected into the mushroom body calyces. These results emphasise the role of the cholinergic networks of the mushroom bodies in the formation and recall of memory in the honeybee. They suggest that the role of the brain structures in these processes is sequential. Mushroom body calyces involved in the associative process of olfactory learning could be relayed by the alpha-lobes for information retrieval.     

The Role of Dopamine in Drosophila Larval Classical Olfactory Conditioning

Learning and memory is not an attribute of higher animals. Even Drosophila larvae are able to form and recall an association of a given odor with an aversive or appetitive gustatory reinforcer. As the Drosophila larva has turned into a particularly simple model for studying odor processing, a detailed neuronal and functional map of the olfactory pathway is available up to the third order neurons in the mushroom bodies. At this point, a convergence of olfactory processing and gustatory reinforcement is suggested to underlie associative memory formation. The dopaminergic system was shown to be involved in mammalian and insect olfactory conditioning. To analyze the anatomy and function of the larval dopaminergic system, we first characterize dopaminergic neurons immunohistochemically up to the single cell level and subsequent test for the effects of distortions in the dopamine system upon aversive (odor-salt) as well as appetitive (odor-sugar) associative learning. Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies. However, a number of dopaminergic neurons innervate different regions of the brain, including protocerebra, mushroom bodies and suboesophageal ganglion. We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants. Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae. Our data suggest that dopaminergic neurons provide input to different brain regions including protocerebra, suboesophageal ganglion and mushroom bodies by more than one route. We therefore propose that different types of dopaminergic neurons might be involved in different types of signaling necessary for aversive and appetitive olfactory memory formation respectively, or for the retrieval of these memory traces. Future studies of the dopaminergic system need to take into account such cellular dissociations in function in order to be meaningful.     

Drosophila dorsal paired medial neurons provide a general mechanism for memory consolidation

Memories are formed, stabilized in a time-dependent manner, and stored in neural networks. In Drosophila, retrieval of punitive and rewarded odor memories depends on output from mushroom body (MB) neurons, consistent with the idea that both types of memory are represented there. Dorsal Paired Medial (DPM) neurons innervate the mushroom bodies, and DPM neuron output is required for the stability of punished odor memory. Here we show that stable reward-odor memory is also DPM neuron dependent. DPM neuron expression of amnesiac (amn) in amn mutant flies restores wild-type memory. In addition, disrupting DPM neurotransmission between training and testing abolishes reward-odor memory, just as it does with punished memory. We further examined DPM-MB connectivity by overexpressing a DScam variant that reduces DPM neuron projections to the MB alpha, beta, and gamma lobes. DPM neurons that primarily project to MB alpha' and beta' lobes are capable of stabilizing punitive- and reward-odor memory, implying that both forms of memory have similar circuit requirements. Therefore, our results suggest that the fly employs the local DPM-MB circuit to stabilize punitive- and reward-odor memories and that stable aspects of both forms of memory may reside in mushroom body alpha' and beta' lobe neurons.     

Mushroom-body memories: an obituary prematurely written?

Studies on insect olfactory learning have established the mushroom bodies as key brain structures for the formation of long-term memory (LTM). Two new neurons in the fly brain are reported now as essential sites for LTM formation, while mushroom bodies are claimed to be unnecessary to this end.     

Behavioral functions of the insect mushroom bodies

New methods of intervention in Drosophila and other insect species reveal that the mushroom bodies are involved in a diverse set of behavioral functions. The intrinsic Kenyon cells (those neurons with projections within the mushroom bodies) house part of the short-term memory trace for odors and are required for courtship conditioning memory. A pair of extrinsic mushroom body neurons (neurons with projections both inside and outside the mushroom bodies) provides a neuropeptide important for 1-hour olfactory memory. In addition, the mushroom bodies are necessary for context generalization in visual learning and for regulating the transition from walking to rest.     

Dopamine-like immunoreactivity in the bee brain

Summary The distribution of dopamine-like immunoreactive neurons is described for the brain of the bee, Apis mellifera L., following the application of a pre-embedding technique on Vibratome sections. Immunoreactive somata are grouped into seven clusters, mainly situated in the protocerebrum. Immunoreactive interneurons have been detected in the different neuropilar compartments, except for the optic lobe neuropils. Strong immunoreactivity is found in the upper division of the central body, in parts of the stalk and in the -lobe layers of the mushroom bodies. A dense network of many immunoreactive fibres surrounds the mushroom bodies and the central body. It forms a number of interhemispheric commissures/chiasmata, projecting partly into the contralateral mushroom body and central body. The lateral protocerebral neuropil contains some large wide-field-neurons. The antennal-lobe glomeruli receive fine projections of multiglomerular dopamine-like immunoreactive interneurons.     

Effect of Radicicol Infusion on the <Emphasis Type="Italic">Src</Emphasis> Tyrosine Kinase Activity of Rat Hippocampus before and after Training in an Inhibitory Avoidance Task

The participation of protein serine/threonine kinases in memory formation and retrieval is well established. In contrast, relatively little is known on the role of protein tyrosine kinases (PTKs). Previous work showed that intra-hippocampal infusion of the Src-PTK inhibitor radicicol inhibits memory acquisition, consolidation, and retrieval of one-trial step-down inhibitory avoidance task. In this study, we investigated the possible interaction between levels of Src-PTK activity in hippocampus and memory acquisition, formation, and retrieval of this task. Radicicol (0.5 μg/ml) was infused into the CA1 region of the hippocampus of rats trained in a one-trial step-down inhibitory avoidance task. Radicicol infused 15 min before training decreased Src-PTK activity, as measured 0, 1.5, and 24 h after training, and impaired memory acquisition of the task. When given immediately after training, there was a decrease in Src-PTK activity 1.5 h, but not 0 or 24 h after training. This treatment depressed memory consolidation. Radicicol infused into CA1 10 min prior to retrieval testing inhibited hippocampal Src-PTK activity, as measured immediately after the test session. The results suggest that Src-PTKs participate in memory acquisition, consolidation, and retrieval processes, but the timing of the role of the enzyme is different in each case.     

Heat Shock during the Development of Central Structures of the Drosophila Brain: Memory Formation in the l(1)ts403 Mutant of Drosophila melanogaster

The structures and functions of many genes are homologous in Drosophilaand humans. Therefore, studying pathological processes in Drosophila, in particular neurogenerative processes accompanied by progressive memory loss, helps to understand the ethiology of corresponding human disorders and to develop therapeutic strategies. It is believed that the development of neurogenerative diseases might result from alterations in the functioning of the heat shock/chaperone machinery. In view of this, we used Drosophila mutant l(1)ts403 with defective synthesis of heat shock proteins for studying learning and memory in a test of conditioned courtship suppression following a heat shock given at different developmental stages. High learning indices were registered immediately and 30 min after training both in the intact controls and in flies subjected to different developmental heat shocks. This indicated normal learning and memory acquisition in the mutant. At the same time, memory retention (3 h after training) suffered to different extent depending on the developmental stage. The remote effects of heat shock given during the formation of the mushroom bodies indicated the important role of this brain structure in the memory formation. The observed memory defects may result from alterations both in mRNA transport and in the functions of molecular chaperones in the l(1)ts403 mutant.     

Male-associated polypeptide (MAP) expression in different compartments of the reproductive system of the mussel Mytilus galloprovincialis: immunocytochemical and Western blot study

The dissociation and maintenance in culture of cells derived from the mushroom bodies of adult crickets (Acheta domesticus) are described. This primary culture was developed in order to investigate maturation and differentiation of mushroom-body cells including Kenyon cells, the major intrinsic interneurons of mushroom bodies, which have been shown to be involved in learning and memory in insects. Three distinct cell types were observed, all identified as neural cells on the basis of their size, morphology and immunocytochemical staining with horseradish peroxidase. These cells appear to correspond to the three cell types observed in vivo: Kenyon cells, ganglion mother cells and neuroblasts. Some cells showed neurite growth, usually with long unipolar processes, occasionally with either bipolar or, more rarely, multipolar processes. Neuronal cell bodies readily formed seals with patch pipettes, allowing stable, whole-cell, patch-clamp electrophysiological recordings. Depolarization of the cell under voltage-clamp resulted in at least two types of outwardly directed potassium currents: a delayed rectifier-type of current that was sensitive to tetraethylammonium, and a cadmium-sensitive current with rapid inactivation. Neither type of current was affected by quinidine, a blocker of potassium currents recorded from pupal honeybee Kenyon cells. Other ionic currents, which have yet to be characterized, were also observed. Received: 30 October 1996 / Accepted: 11 July 1997     

Sequential use of mushroom body neuron subsets during drosophila odor memory processing

Drosophila mushroom bodies (MB) are bilaterally symmetric multilobed brain structures required for olfactory memory. Previous studies suggested that neurotransmission from MB neurons is only required for memory retrieval. Our unexpected observation that Dorsal Paired Medial (DPM) neurons, which project only to MB neurons, are required during memory storage but not during acquisition or retrieval, led us to revisit the role of MB neurons in memory processing. We show that neurotransmission from the alpha'beta' subset of MB neurons is required to acquire and stabilize aversive and appetitive odor memory, but is dispensable during memory retrieval. In contrast, neurotransmission from MB alphabeta neurons is only required for memory retrieval. These data suggest a dynamic requirement for the different subsets of MB neurons in memory and are consistent with the notion that recurrent activity in an MB alpha'beta' neuron-DPM neuron loop is required to stabilize memories formed in the MB alphabeta neurons.