Fridericia nanningensis, a new terrestrial enchytraeid species (Oligochaeta) from southwestern China

Fridericia nanningensis, a new species from wetland soil of Nanhu Park, Nanning city, the capital of Guangxi Zhuang Autonomous Region in southwest China, is described. It is characterized by 2-4 chaetae per bundle, poorly-developed clitellar glands, slender, unbranched peptonephridia, and spermathecae with 2 ampullar diverticula, a deep constriction in the middle of the ampulla and one large ectal gland. It is closely related to the European species, F. alata Nielsen & Christensen, 1959 and the East European species, F. tubulosa Dozsa-Farkas, 1972 by the shape of peptonephridia and the undeveloped clitellar glands. It differs from F. alata by its shorter body length and fewer chaetae per bundle, its type of coelomocytes (type "c"), its deep constriction in the middle of the spermathecae ampulla and a larger ectal gland, and it differs from F. tubulosa by its pale epidermal glands, its more anterior origin of the dorsal vessel, a deep constriction in the middle of the spermathecae ampulla, shorter ectal duct, and only one ectal gland at the spermathecal orifice.

Two new species of Rotifera from China

Two new rotifera species, Brachionus huangi n. sp, and Lecane chinesensis n. sp. are described and illustrated from different localities in China. The main distinguishing taxonomic features of B. huangi are three pairs of apical spines, a pair of symmetrical caudal spines and a wide caudal projection. For L. chinesensis, the main characteristics are the two-sided small extentions of the foot pseudosegment, the long and innercurved apical spines and ventral lorica anteriorly narrower than the dorsal one.

海马在情绪记忆中的作用

海马是中脑边缘系统的一个重要区域，不仅在学习和记忆中起着重要作用， 还在应激调控中起着关键作用。越来越多的报道表明，海马可能参与毒品成瘾。 本文围绕海马与成瘾、海马与应激的相互关系，应用行为学及电生理技术手段探 讨了海马在成瘾中的作用，并对应激的作用机制进行了研究。 第一部分试验首先研究了长期成瘾药物暴露对两种不同类型学习记忆的影 响，从而揭示成瘾对不同脑区的影响；我们还研究了长期毒品戒断对学习和记忆 的影响。用条件化位置偏爱/厌恶模型对海马在成瘾相关记忆中的作用进行了探 讨。实验结果提示，在成瘾过程中不同脑区发生了不同程度的适应性改变，从而 影响了该脑区相关的正常学习和记忆，其中慢性吗啡暴露损伤了海马依赖的空间 记忆提取，但长期戒断（戒断5 个星期）可以缓解成瘾导致的这些影响。另外， 海马在成瘾相关记忆中同样发挥了重要作用。吗啡偏爱形成后的环境线索再暴露 损伤海马突触长时程增强，提示了海马在环境线索诱导的药物渴求和复吸中可能 起着关键性作用。 第二部分试验主要探索了应激效应的作用机制及海马VR1 受体对抗应激效 应。发现含NR2B 的NMDA 受体参与了应激导致的海马突触可塑性改变，其阻 断剂Ro25-6981 阻断了应激易化的海马长时程抑制，避免了应激损伤的长时程增 强。另一方面，应激损伤海马依赖性的空间记忆提取，但是辣椒素(VR1 受体的 激动剂)直接注射入海马能逆转这种应激的损伤效应。相反，VR1 受体拮抗剂 capsazepin 处理产生了类似应激的损伤效应。提示了海马内VR1 受体可能是海马 记忆功能的一种重要受体系统。

1. 大鼠海马-前额叶回路在学习记忆中的作用2. 芬克罗酮改善恒河猴空间工作记忆的谷氨酸机制

一、大鼠海马-前额叶回路在学习记忆中的作用 解剖学研究证实大鼠和猴的海马结构（hippocampal formation, HF；本文‘海马 (hippocampus, Hip)’一词即指海马结构）和前额叶 (prefrontal cortex, PFC) 之间存在一条单向、同侧和单突触的神经回路，即海马-前额叶回路（Hip-PFC回路）。Hip和PFC均参与学习记忆等多种认知功能，PFC是工作记忆的关键脑区，而Hip是空间参考记忆的关键脑区。虽然人们已经对PFC和Hip进行了广泛深入的研究，但对Hip-PFC回路参与哪些认知功能还知之甚少。本研究的目的就是通过暂时阻断Hip-PFC回路，探讨其在学习和记忆中的作用。 在大鼠，Hip-PFC回路中的纤维主要从Hip腹部 (ventral hippocampus, VH)发出，投射到PFC的前边缘皮质(prelimbic cortex, PLC)、下边缘皮质 (infralimbic cortex, ILC) 和外侧前额叶 (lateral prefrontal cortex) 等亚区，其中PLC是Hip-PFC主要投射的区域。我们通过给动物安装慢性导管向脑内注射GABAA受体激动剂muscimol (MU) 阻断Hip-PFC回路。注射位点包括 ①双侧PLC，②双侧VH，③一侧VH和对侧PLC (VH-PLC)。我们首先观察了在PLC或VH局部注射MU对自由活动大鼠PLC和VH脑电功率的影响，并以此确定在行为实验中所用蝇蕈醇的剂量。然后采用T-迷宫空间交互延缓作业 (spatial delayed alternation task) 测试Hip-PFC回路被阻断的动物的空间工作记忆功能；采用被动回避作业 (passive avoidance task) 测试其情绪相关记忆的能力（训练前给药；24 h后重测试）；采用Morris水迷宫作业 (Morris water maze task) 测试其空间参考记忆的能力（每天训练前给药；训练期（3 d）结束24 h后重测试）。结果表明：在大鼠PLC或VH局部注射0.5 μg/0.25μl MU后30 min显著抑制VH 和PLC的脑电功率 (VH, p < 0.01; PLC, p < 0.05 vs. PBS/baseline)。注射MU (0.5 μg/0.25μl) 到 ①双侧PLC、②双侧VH、③VH-PLC均显著降低动物在空间交互延缓作业 (All p < 0.001, vs. PBS) 和空间Morris水迷宫作业中的成绩 (All p < 0.05, vs. PBS)，表明Hip-PFC回路在空间工作记忆（空间短时记忆）和在空间参考记忆（空间长时记忆）中均起重要作用。在空间交互延缓作业中，双侧PLC被抑制的大鼠的成绩显著低于双侧VH或VH-PLC被抑制的动物，说明PFC在空间工作记忆功能中占有主导地位。在被动回避作业中，双侧VH被抑制动物的回避反应的潜伏期显著短于对照动物 (p < 0.05 vs. PBS)，说明双侧VH被抑制动物的情绪记忆受损；而双侧PLC或VH-PLC被抑制的动物其回避反应的潜伏期与对照动物无显著差异 (PLC, p > 0.9; VH-PLC, p > 0.3 vs. PBS)，表明双侧PLC或VH-PLC被抑制的动物情绪记忆正常。被动回避作业的结果说明VH参与情绪记忆的形成，但Hip-PFC回路在情绪记忆形成中不起重要作用。 以上结果表明，大鼠Hip-PFC回路参与空间工作记忆和空间参考记忆而不是情绪记忆功能。情绪记忆的关键脑结构是杏仁复合体 (amygdala complex, AMC)，VH与AMC有密切的纤维联系。VH被抑制的大鼠情绪记忆受损，说明情绪记忆可能与AMC-Hip回路有关。情绪记忆与空间记忆（参考记忆和工作记忆）在解剖上的分离说明，对于不同类型的记忆来说，其在脑内的信息加工过程是并行的。神经回路内部的信息加工过程则是串行的，回路上任何一个结构的破坏均可导致回路功能的损伤。本研究的结果为学习记忆的“多重记忆系统”理论和记忆信息加工的串行并行机制提供了新的实验证据。 二、芬克罗酮改善成年恒河猴空间工作记忆的谷氨酸机制 芬克罗酮是中科院昆明植物所郝小江等合成的取代吡咯烷酮类化合物。中科院昆明动物所蔡景霞等发现芬克罗酮能改善东莨菪碱、育亨宾等导致的多种动物的不同类型的学习记忆障碍，提高老年动物的学习记忆能力，尤其是老年猴的空间工作记忆。已证实芬克罗酮为部分钙激动剂，可使脑缺血沙土鼠脑内升高的谷氨酸降低，而使正常的沙土鼠海马胞外谷氨酸释放增加。那么芬克罗酮能否提高正常动物的学习记忆，其对正常动物学习记忆的提高是否与其增加谷氨酸的释放有关？本研究采用空间延缓反应作业和谷氨酸NMDA受体拮抗剂MK-801在正常成年猴恒河猴上探讨了以上问题。 结果表明，口服芬克罗酮可显著提高成年猴的空间工作记忆，其量效曲线呈倒‘U’形，符合许多促智药的量效特点。0.25 mg/kg和0.5 mg/kg为芬克罗酮的最佳有效剂量 (p < 0.05 vs. 安慰剂)。肌注MK-801 (0.1 mg/kg) 显著降低成年猴的空间工作记忆 (p < 0.01 vs. 安慰剂)，而口服2.0 mg/kg和4.0 mg/kg的芬克罗酮则显著改善MK-801导致的工作记忆障碍 (p < 0.05 vs. MK-801)。芬克罗酮的所有测试剂量不影响猴在作业中的反应时 (p > 0.05 vs. 安慰剂)，表明芬克罗酮在该剂量范围不影响动物的运动能力。 本研究结果提示，芬克罗酮可能通钙激动作用促进谷氨酸的释放，在一定剂量范围内提高胞外谷氨酸水平，提高正常动物的空间工作记忆等认知功能。 关键词：芬克罗酮，恒河猴，空间工作记忆，空间延缓反应作业，谷氨酸，MK-801

角蟾科（Megophryidae; Anura）蝌蚪的形态多样性和生态适应

角蟾科（Megophryidae）是以角蟾属（Megophrys Kuhl and Van Hasselt, 1822）为模式属而建立的，隶于无尾目（Anura），变凹型亚目（Anomocoela）。角蟾科包括2 亚科11 属142 种，分布于东洋界，从巴基斯坦、中国西部向东直到菲律宾和苏达群岛；中国有9 属75 种分布于华中和华南地区。角蟾科被认为是原始的两栖动物之一，其分类学、系统学、生态学、动物地理学的研究均深受中外科学家的瞩目。近年来，通过形态学、古生物学、细胞学、生态学、支序系统学的研究，角蟾科的分类与系统学研究取得了较大进展。与成体形态和分子系统学研究结果相比较，蝌蚪的研究存在更多的问题和挑战，尚需深入研究：（1）角蟾科蝌蚪的形态多样性分析；（2）角蟾科的系统发育关系与蝌蚪的演化，以及口漏斗的起源；（3）角蟾科蝌蚪表型分化与栖息环境和觅食行为的适应演化。针对上述问题，本文对角蟾科9 属30 种蝌蚪的形态特征，包括外部宏观形态和口器外部结构特征、口器内部显微结构、唇齿和角质颌的亚显微结构作了深入细致、多层次的比较研究；通过12s rRNA 和cytochrome b 基因构建最大简约树，采用贝叶斯系统发育进行分析，蝌蚪型的演化采用祖先性状的重建方法分析；得到如下结论：1）初步将角蟾科蝌蚪分为4 种类型；并且建立了2 种新的角蟾科蝌蚪类型。A 型：拟髭蟾型蝌蚪，该型蝌蚪包括拟髭蟾属、髭蟾属、齿蟾属和齿突蟾属的物种；B 型：新类型，掌突蟾型蝌蚪，该型蝌蚪在本文中包括掌突蟾属、小臂蟾属的物种；C 型：新类型，短腿蟾型蝌蚪，一种特化类型，该型蝌蚪在本文中仅包括短腿蟾属的物种；D 型：角蟾型蝌蚪，该型蝌蚪在本文中包括无耳蟾属、小口拟角蟾属和异角蟾属的物种。2）对角蟾科的分类进行了修订：（1）支持角蟾科两个亚科的分类系统；（2）角蟾亚科包括拟角蟾属、异角蟾属、无耳蟾属和短腿蟾属；该亚科形态差异小，系统学关系比较复杂，暂不作族级分类的再划分；（3）拟髭蟾亚科分为2 个族：拟髭蟾族，该族物种具有类型A 的蝌蚪，包括4 个属：拟髭蟾属、髭蟾属、齿蟾属、齿突蟾属；掌突蟾族，该族物种具有类型B 的蝌蚪，包括2 个属：掌突蟾属和小臂蟾属。3）结合分子系统进化关系探讨了4 种蝌蚪类型的演化。（1）角蟾科蝌蚪的最近共同祖先来自于一类具有拟髭蟾型蝌蚪性状的蝌蚪；（2）掌突蟾型蝌蚪和角蟾亚科的蝌蚪是由具有拟髭蟾型蝌蚪性状的祖先蝌蚪分别演化而来；（3）短腿蟾型蝌蚪是角蟾型蝌蚪的一种特化类型；（4）外群蝌蚪具有与拟髭蟾型蝌蚪相似的性状，进一步印证了类拟髭蟾型蝌蚪是角蟾科蝌蚪的最近共同祖先的假说；（5）具有口漏斗的蝌蚪类型是由不具口漏斗的蝌蚪类型演化而来，在角蟾科中口漏斗是一种衍生性状。4）分析了角蟾科四种蝌蚪类型与栖息环境的适应演化。（1）角蟾科蝌蚪的口部和体形的变化反映了该科蝌蚪由缓流向类似静水生境的回水凼的渐变式适应，角蟾科蝌蚪的形态显示了多方面的适应变化；（2）随着蝌蚪类型由A 向D的演化，当水速较大时，拟髭蟾型的蝌蚪营流水攀吸型生活方式；当水速递减时，掌突蟾型蝌蚪营流水附着型生活方式；当水速进一步递减时，具有较小口漏斗的短腿蟾型蝌蚪和具有大漏斗的角蟾型蝌蚪营流水浮泳型生活。角蟾科蝌蚪对于水流递减的适应演化说明蝌蚪的生态学适应是具有进化意义的；（3）蝌蚪口器内部结构的分化揭示了蝌蚪和食性的适应关系，蝌蚪以口部的唇齿与角质颌刮取或吞吸水中的物质，然后，通过口乳突有选择地过滤进入口腔中食物。拟髭蟾亚科蝌蚪的唇齿多而窄，唇齿间距宽，颌鞘粗而稀，反映了其植食性为主的特点；它们的舌前乳突一般为指状，在口腔入口处所占面积小，其机械过滤的作用很多被唇齿和角质颌分担了；而角蟾亚科的蝌蚪，其角质颌弱，其舌前乳突一般为匙状，几乎填满了口腔入口处，因此舌前乳突起了主要的机械过滤作用。The family Megophryidae is the largest and most diverse families inArchaeobatrachia, and most of its species occur in India, Pakistan, and eastward intoChina, Southeast Asia, Borneo and the Philippines to the Sunda Islands. Currently thefamily includes 142 species have been grouped into two subfamilies, Megophryinaeand Leptobrachiinae. The mountains of central and southern China are rich in speciesof Megophryidae, 75 species belong to 9 genera and two subfamilies.The family was supposed to be ideal materials of studies in many fields of biology,such as taxonomy, evolution, systematics, ecology, and biogeography. Recently, therehave a great development in taxonomy and systematics of megophryids throughstudied by morphology, paleontology, cytology, ecology, and cladistics. However,larvae of megophryids were generally unknown, although the tadpoles might be veryimportant for above studies.In this paper, we examined the evolutionary scenario of the tadpoles’ morphologyin the context of a phylogenetic framework. Our objectives are (1) to evaluate thedivergence of larval body shape and oral discs in the family Megophryidae, (2) toexplore the evolutionary trends of the larvae in megophryidae, and test if thefunnel-shaped oral disc is apomorphic, and (3) to explore the relationship of the larvalstructure, diet and microhabitat.We examined larval morphology of 30 megophryid species, the larval body shape,oral discs, the buccopharyngeal cavity, and jaw sheaths and denticles of the Chinesemegophryid frogs were re-examined. We constructed a phylogeny of the species on thebasis of published mitochondrial cytochrome b and 16S rRNA gene segments usingpartitioned Bayesian analyses. Furthermore, hypothetical changes of larval morphologywere inferred using parsimony principle on the phylogeny. The results showed that:1) Four tadpole types in Megophryidae. The larval morphological charactersseries in Chinese megophryids fall into four general categories according to the bodyshape and oral discs: (A) Leptobrachiini type, species from genera Leptobrachium,Oreolalax, Scutiger and, Vibrissaphora share this type of tadpoles. (B) Leptolalax type,species of genus Leptolalax have this type of tadpoles. (C) Brachytarsophrys type,species of the genus Brachytarsophrys have this type of tadpoles. (D) Megophryinitype, species of the genera Atympanophrys, Ophryophryne, and Xenophrys share this type of tadpoles. Of which B and C are two novel types.2）Taxonomic implications. The present study leads us to reconsider the generalclassification of tribes attributed to members of Megophryidae. More specifically,concerning the phylogenetic relationships and the two novel tadpole types describedherein, we propose a provisional taxonomy for the family but suggest that further taxasampling of other megophryids be performed to confirm this taxonomic change. TheMegophryidae is composed of two subfamilies (Leptobrachiinae and Megophryinae).The Leptobrachiinae was recogonized the two tribes: (1) tribe Leptobrachiini sensuDubois, corresponding to the tadpole of type A, including four genera, i.e.,Leptobrachium, Oreolalax, Scutiger and, Vibrissaphora; (2) tribe Leptolalaxini,corresponding to the tadpole of novel type B, including two genera, i.e., Leptolalaxand Leptobrachella. However, the relationships among the genera of Megophryinaewere largely unresolved, they recognized no monophyletic groups above the generalevel. A more thorough sampling will likely foster a better taxonomic solution.3) The larval evolutionary scenario in Megophryidae.Type A is characteristicof normal-mouthed with multiple tooth rows, representing the tadpole type of theMRCA of Chinese megophryids. Type B is characteristic of normal-mouthed withreduced tooth rows, prolonging labium, and integumetary glands. Type C ischaracteristic of no labial teeth and smaller umbeliform oral disc. Type D ischaracteristic of no labial teeth, enlarged umbeliform oral disc, representing the tadpoleof the MRCA of subfamily Megophryinae. A previous hypothesis, referring tofunnel-shaped oral discs as an apomorphy, is supported.4) The larval adaptation to habitats in Megophryidae. Tadpoles generallyadhere to substrates using their mouths, and the microhabitat that the tadpoles occupyreflects the degree of adhesion and oral complexity. The morphological changes inmegophryid tadpoles virtually allow a progressive adaptation to a changing habitatfrom faster water to slower water. Within the tadpoles of Type A to type D, the TOTbecomes smaller and smaller, and the oral disc orientates from anteroventral toumbelliform upturned, and eye position orientates from dorsal to lateral, and the trunkis more and more depressed and tail becomes relatively longer and slender. Within therunning water, the normal-mouthed with multiple tooth rows of Leptobrachiini tadpoles are correlated with lotic-suctorial, benthic feeders with anteroventral oraldisc and the largest body. With the water’s velocity decreasing, the lotic-adherentfeeders of Leptolalax tadpoles have tube-shaped labium with reduced tooth rows andintegumetary glands. And then, the smaller umbeliform in Brachytarsophrys tadpolesand the enlarged umbeliform oral disc in the Megophryini tadpoles are inhabitmicrohabitats of non-flowing backwaters of rivers, indicative of adaptive traits oflotic-neustonic surface feeders. The scheme of megophryid tadpoles andmicrohabitats provided the first clear evidence which congruent with the hypothesis ofAltig and Johnston (1989). The ecological divergence plays a general role in thedivergence and evolution of megophrid larvae. There is a definite correlation amongthe buccopharyngeal cavity, diet and feeding mechanisms, the tadpole graze orswallow the food particles, then through papillae which like a sieve and sort out foodparticles to the oesophagus. The tadpole of Leptobrachiinae possess multiple toothrows, wide intertooth distance as well as thick and sparse jaw sheath, these tadpolesinhabit bottom of the streams and graze on epiphyton or major detritus of organicmatter on the substrates, their prelingual papillae like single finger, the mechanicalpurpose of papillae served share in by tooth and jaw. The tadpoles of Megophryinaeoccur near the water surface of small streams and are the filter feeder, their dietincludes plankton and organic debris floating on the water surface, those tadpolepossess weak jaw, their prelingual papillae like spoon, the mechanical purpose ofpapillae served mostly for sieve.

Effects of Naotan Pill on repair of neural cells and cognitive disorders in juvenile rats following hypoxia and ischemia

BACKGROUND: Hypoxia and ischemia induce neuronal damage, decreased neuronal numbers and synaptophysin levels, and deficits in learning and memory functions. Previous studies have shown that lycium barbarum polysaccharide, the most effective component of barbary wolfberry fruit, has protective effects on neural cells in hypoxia-ischemia. OBJECTIVE: To investigate the effects of Naotan Pill on glutamate-treated neural cells and on cognitive function in juvenile rats following hypoxia-ischemia. DESIGN, TIME AND SETTING: The randomized, controlled, in vivo study was performed at the Cell Laboratory of Lanzhou University, Lanzhou Institute of Modern Physics of Chinese Academy of Sciences, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from December 2005 to August 2006. The cellular neurobiology, in vitro experiment was conducted at the Institute of Human Anatomy, Histology, Embryology and Neuroscience, School of Basic Medical Sciences, Lanzhou University, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from March 2007 to January 2008. MATERIALS: Naotan Pill, composed of barbary wolfberry fruit, danshen root, grassleaf sweetflag rhizome, and glossy privet fruit, was prepared by Gansu Provincial Rehabilitation Center, China. Rabbit anti-synaptophysin, choline acetyl transferase polyclonal antibody, streptavidin-biotin complex kit and diaminobenzidine kit (Boster, Wuhan, China), as well as glutamate (Hualian, Shanghai, China) were used in this study. METHODS: Cortical neural cells were isolated from neonatal Wistar rats. Neural cell damage models were induced using glutamate, and administered Naotan Pill prior to and following damage. A total of 54 juvenile Wistar rats were equally and randomly assigned into model, Naotan Pill, and sham operation groups. The left common carotid artery was ligated, and then rat models of hypoxic-ischemic injury were assigned to the model and Naotan Pill groups. At 2 days following model induction, rats in the Naotan Pill group were administered Naotan Pill suspension for 21 days. In the model and sham operation groups, rats received an equal volume of saline. MAIN OUTCOME MEASURES: Neural cell morphology was observed using an inverted phase contrast microscope. Survival rate of neural cells was measured by MTT assay. Synaptophysin and choline acetyl transferase expression was observed in the hippocampal CA1 region of juvenile rats using immunohistochemistry. Cognitive function was tested by the Morris water maze. RESULTS: Pathological changes were detected in glutamate-treated neural cells. Neural cell morphology remained normal after Naotan Pill intervention. Absorbance and survival rate of neural cells were significantly greater following Naotan Pill intervention, compared to glutamate-treated neural cells (P < 0.05). Synaptophysin and choline acetyl transferase expression was lowest in the hippocampal CA1 region in the model group and highest in the sham operation group. Significant differences among groups were observed (P < 0.05). Escape latency and swimming distance were significantly longer in the model group compared to the Naotan Pill group (P < 0.05). CONCLUSION: Naotan Pill exhibited protective and repair effects on glutamate-treated neural cells. Naotan Pill upregulated synaptophysin and choline acetyl transferase expression in the hippocampus and improved cognitive function in rats following hypoxia-ischemia.

Fruit anatomy,seed germination and seedling development in the Japanese seagrass phyllospadix

Phyllospadix iwatensis Makino and phyllospadix japonicus Makino have similar frunt morphology and anatomy.The rhomboid fruit of Japanese phyllospadix is dark brown in colour and is characterized by two arms bearing stiff inflected bristles which can act as an anchoring system. The fruit covering consists of a thin cuticular seed coat and pericarp remains mainly fibrous endocarp. In the groove region of the fruit.the cuticular seed coat and endocarp are replaced by nucellus cells with wall in growths and crushed pigment strands with lignified walls.these tissues appera to control the transfer of nutrients to developing seed.the seed is oval with a small embryo and a large hypocotyl. the embryo is straight and simple,with the plumule containing three leaf primordia and a pair of root primordia surrounded by a cotyledon.the hypocotyl has large vontral lobe containing central provascular tissue and two small dorsal lobes.the hypocotyl contains starch.lipid and protein.and acts as a nutrient store.the seed of P.iwatensis has a dormancy period of 2-6 weeks and germination eventually reaches-65%.but is not synchronized.during germination the leaves emerge first.and then after at least three young leaves have formed and abseised.the roots emerge,usually?6 months after the commencement of germination.Utilizaton of the nutrient reserves is initially from the perihpery of the hypocotyl and then progressively towards its centre.

抑制前额叶和海马的星形胶质细胞功能对大鼠工作记忆的影响，长期服用芬克罗酮改善老年大鼠学习记忆障碍的突触可塑性机制

长期以来，星形胶质细胞（astrocyte，AS）被认为具有细胞隔离、支持、保护和营养神经元的作用。近来，人们逐渐发现，AS还有许多其他重要的功能。例如，谷氨酸是中枢神经系统中重要的兴奋性神经递质，AS摄取和合成谷氨酸的作用可能影响学习、神经发育及发情周期等多种脑功能，并影响突触形成和突触传递效能。有研究发现，成年海马齿状回颗粒下层具有神经干细胞潜能的AS，可以分化成新生神经元。此外，海马成熟的AS还参与调节神经发生，决定神经干细胞的分化方向。大鼠海马新生神经元与海马依赖性记忆（happocampal- dependent memory）密切相关。提示，AS与学习记忆密切相关。但迄今为止，尚未见AS参与工作记忆的报道。本研究以特异性抑制AS细胞的化合物fluorocitrate（FC）作为工具药来探讨AS在大鼠工作记忆中的作用及作用机制。FC可抑制AS三羧酸循环中乌头酸酶（aconitase）的活性，从而抑制AS摄取谷氨酸的功能，其作用是可逆的，目前已经被广泛用于研究AS的功能。我们通过慢性导管在大鼠双侧前额叶（prefrontal cortex，PFC），双侧海马（hippocampus，HIP）和单侧侧脑室（lateral ventricle，LV）分别注射FC，记录注射后15-75min内自由活动大鼠的脑电图（EEG）功率变化，以判断FC对大鼠PFC和HIP神经元兴奋性的影响，并在注射后15-75min内完成T-迷宫空间交互延缓(spatial delayed alternation task)反应测试，分析动物的空间工作记忆功能。结果发现在双侧PFC和双侧HIP分别注射1，2，5nM FC （0.5μl），仅2，5nM显著增加HIP EEG的功率（ p < 0.05 vs. SAL），但对交互延缓反应成绩无明显影响，所有剂量对PFC的EEG功率没有显著影响，且不影响动物操作交互延缓反应作业的成绩（ALL p > 0.1 vs. SAL）。在单侧LV注射0.05，0.5nM FC （10μl）后，PFC的EEG功率没有明显变化，而HIP的EEG功率有增加。该剂量的FC对大鼠操作空间交互延缓作业的成绩无明显影响。在单侧LV注射2nM FC（10μl）同时显著增加PFC和HIP的EEG功率，并显著提高大鼠操作空间交互延缓反应作业中的正确率，改善大鼠的工作记忆（ p < 0.05 vs. SAL）。单侧LV注射5nM FC（10μl）同时显著降低PFC和HIP的EEG功率（ALL p < 0.05 vs. SAL）和大鼠操作作业的正确率，损伤大鼠的工作记忆（ p < 0.05 vs. SAL）。表明只有在PFC和HIP的EEG功率同时受到影响的时候，大鼠操作作业的正确率才会发生显著改变。已有研究指出，前额叶和海马均参与工作记忆调控。在LV给FC将同时作用于双侧PFC和双侧HIP。因此，我们认为，FC对大鼠空间交互延缓作业成绩的影响可能是通过抑制PFC和HIP的AS功能产生的。在单侧LV注射低浓度（0.05，0.5nM）的FC对大鼠空间交互延缓作业成绩和EEG功率水平均无明显影响，可能与脑内AS的功能尚可，谷氨酸水平升高不明显有关，而高浓度（5nM）FC可能较强地抑制AS摄取胞外谷氨酸的功能，胞外谷氨酸水平过度升高，损伤神经元的活性，使EEG功率降低，结果导致工作记忆受损。高浓度FC也可能直接损伤神经元的活性。本研究结果表明，FC短期抑制PFC和HIP的AS功能，将使PFC和HIP胞外谷氨酸水平升高，从而改变PFC和HIP神经元的兴奋性，使大鼠的空间工作记忆功能发生改变。揭示了工作记忆不仅依赖于PFC-HIP回路中神经元的正常功能，也与PFC-HIP回路中AS的功能正常与否密切相关。

1、吗啡成瘾及吗啡对其他学习记忆的影响 2、老年猕猴初级视觉皮层细胞时间及空间特性的改变

一、 药物滥用是一种慢性、复发性脑疾病。药物滥用将导致药物成瘾（addiction），其主要表现有药物依赖、药物耐受、药物敏感化以及药物停用后的戒断症状（withdraw symptom）。药物成瘾的核心特征是强迫性觅药和用药行为。药物成瘾会导致药物滥用者认知功能的损伤和认知偏差，并会造成滥用者情绪异常。药物成瘾是一个复杂的生物学过程，有着及其复杂的机理。对药物成瘾机制的解释有很多种，主要认为成瘾过程是一种学习记忆过程，学习记忆的机制在药物成瘾过程中起到了非常重要的作用。首先，学习记忆和药物成瘾过程都受到了相似的神经营养因子以及神经递质系统的调控，例如：它们都受cAMP，CREB等调控因子的调控。其次，研究发现与成瘾相关的线索，如用药有关的人物、地点或暗示等，在药物戒断很长时间后都会恢复吸毒者的用药行为。并且，当把与成瘾相关的线索呈现给毒品戒断中的人时，这些人会出现心率、呼吸加快，血压升高等现象，甚至表现出明显的渴求行为。药物对学习记忆的影响是复杂的，虽然重复使用药物会导致药物成瘾，并且这个过程需要学习记忆机制的参与，但同时使用吗啡却会对其他类型的学习记忆（如：恐惧性学习记忆、一次性被动回避学习记忆和水迷宫空间学习记忆）造成破坏。学习前给予吗啡可以剂量及状态依赖地破坏被动回避试验以及空间辨别试验的记忆获取过程。学习过程结束后立即给予吗啡可以破坏一次性被动回避试验、主动回避试验和恐惧条件化试验的记忆巩固过程。测试前给予吗啡可以破坏空间辨别试验的记忆提取过程。本研究的目的在于更进一步地了解使用吗啡导致吗啡成瘾以及使用吗啡导致学习记忆的各个阶段受损的机制。为此我们采用了药理学以及多种行为学的方法，1、用PTZ诱发的癫痫持续状态干扰吗啡成瘾的学习记忆过程，进一步比较了吗啡成瘾的学习记忆与其他学习记忆，例如：空间学习记忆以及食物奖赏学习记忆的机制有何异同；2、研究了β-肾上腺素系统与阿片系统在空间记忆巩固过程中的相互作用；3、我们还研究了NMDA受体的激动剂和拮抗剂在吗啡破坏空间记忆提取过程中的作用。研究结果发现： 1．戊四唑诱发的癫痫持续状态，对吗啡建立的条件化位置偏好没有任何影响，动物仍然对阳性箱（吗啡匹配箱）表现出明显的偏好。但是癫痫持续状态破坏了食物建立的条件化位置偏好，并且还破坏了水迷宫和Y迷宫检测的空间记忆。癫痫持续状态破坏了食物建立的条件化位置偏好，原因不是由于其影响了动物的食欲。此外，癫痫持续状态也没有持续地破坏动物的活动能力，因此，对动物活动量的影响也不是造成其他学习记忆破坏的原因。这些结果说明，吗啡成瘾的学习记忆和普通的学习记忆在机制上可能存在不同之处。为了说明这个问题，我们还需要进行其他更深入的研究。 2、训练后立即单独注射吗啡（0.25和2.5 mg/kg）或心得安（2，10和20 mg/kg）都不会破坏动物Y-迷宫空间记忆的巩固过程，动物仍然能识别新异环境，并在里面停留较长时间。但是，训练后同时注射吗啡和心得安却可以破坏动物空间记忆的巩固过程。并且，较高剂量的吗啡（2.5 mg/kg）加上较高剂量的心得安（10和20 mg/kg）对记忆的破坏更严重，实验组动物在新异环境停留的时间显著低于对照组。这说明阿片系统和去甲肾上腺素系统在破坏记忆巩固的过程中可能有协同作用。 3、记忆提取前30分钟注射吗啡（1和10 mg/kg）可以剂量依赖地破坏Y-迷宫空间记忆的提取。单独注射NMDA受体的激动剂NMDA（1，2和4 mg/kg）对动物的空间记忆提取没有影响，但是，单独注射NMDA受体拮抗剂MK-801（0.05，0.1和0.2 mg/kg）剂量依赖地破坏了空间记忆的提取。同时注射吗啡（10 mg/kg）和NMDA（2 mg/kg）可以阻断吗啡对空间记忆造成的破坏作用。相反，共同注射吗啡（1 mg/kg）和MK-801（0.05 mg/kg）可以加重吗啡对空间记忆造成的破坏作用。这说明谷氨酸系统可以干扰吗啡对记忆提取过程的影响。 二、衰老严重地影响了人们的视觉功能，然而眼睛光学系统的老年性改变并不能完全解释清楚这种视觉功能衰退。一般认为是神经系统的退化导致了这种老年性功能降低。但是，研究显示视网膜（retina）和外膝体（dorsal lateral geniculate nucleus, dLGN）在衰老的过程中神经元的数量和体积以及神经元的功能特性，如对比度敏感性、空间分辨率等，都没有明显的变化，因此，人们推测老化导致的神经系统的变化发生在更高级的视觉皮层。过去几年的研究发现老年动物视觉皮层细胞发生了一系列反应特性的改变，如：老年动物皮层细胞的方向选择性和方位选择性降低以及细胞反应的潜伏期延长。这些细胞水平的变化被认为是老年性视觉功能衰退的神经机制。为了更全面地了解衰老过程对视觉皮层的影响以及细胞反应改变与整体功能降低之间的关系，本研究采用活体动物细胞外单位记录的方法，比较了青年和老年猕猴初级视觉皮层细胞时间反应特性和空间反应特性的差异。研究结果发现：老年动物初级视觉皮层细胞的时间频率和空间频率敏感性明显比年轻动物降低。表现为老年动物初级视觉皮层细胞的最优时间和空间频率、空间分辨率（spatial resolution, SR）和较高时间截至频率（high temporal frequency cut-off, TF50）都显著低于年轻动物初级视觉皮层细胞，同时伴随着这些功能的降低，老年动物初级视觉皮层细胞的自发放增加，对视觉刺激的反应增加，但是信噪比却显著降低。这些结果表明，老年动物初级视觉皮层细胞的功能在老化过程中都普遍降低。这可能是导致老年人视觉功能降低的原因。

Time-dependent DNA condensation induced by amyloid beta-peptide

The major protein component of the amyloid deposition in Alzheimer's disease is a 39-43 residue peptide, amyloid beta (A beta). A beta is toxic to neurons, although the mechanism of neurodegeneration is uncertain. Evidence exists for non-B DNA conformation in the hippocampus of Alzheimer's disease brains, and A beta was reportedly able to transform DNA conformation in vitro. In this study, we found that DNA conformation was altered in the presence of A beta, and A beta induced DNA condensation in a time-dependent manner. Furthermore, A beta sheets, serving as condensation nuclei, were crucial for DNA condensation, and Cu2+ and Zn2+ ions inhibited A beta sheet-induced DNA condensation. Our results suggest DNA condensation as a mechanism of A beta toxicity.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Expression of Bblhx3, a LIM-homeobox gene, in the development of amphioxus Branchiostoma belcheri tsingtauense

Amphioxus Bblhx3 was identified as a LIM-homeobox gene expressed in gastrulae. Structural analysis suggested that it is a member of lhx3 but not of lhx1 gene group. Whole mount in situ hybridization revealed, that expression of Bblhx3 was initiated at the early gastrula stage and continued at least until 10-day larvae. Expression of Bblhx3 first appeared in the vegetal and future dorsal area in initial gastrulae and became restricted to the endoderm during gastrulation. In neurulae and early larvae, Bblhx3 was expressed in the developing neural tube, the notochord and preoral pit lineage. In 10-day larvae, Bblhx3 was expressed only in the preoral pit. This expression pattern is apparently distinct from that of vertebrate lhx3 genes that are not expressed during gastrulation. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

beta-catenin in early development of the lancelet embryo indicates specific determination of embryonic polarity

The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.

Early expressed genes showing a dichotomous developing pattern in the lancelet embryo

Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3 beta class genes The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region. respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling, The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.

中国明对虾核转录因子NF-κB家族基因的克隆与表达分析

对虾流行病爆发以来，我国乃至世界的对虾产业一直受到各种虾病的困扰，对虾养殖业严重受阻。解决这一问题的关键是加强对虾免疫机制的研究，并在此基础上寻找对虾疾病防治的有效方法。Rel/NF-κB是一类核转录因子，在无脊椎动物的先天性免疫中，起着极为重要的作用。 本论文根据其他无脊椎动物中NF-κB家族基因Relish和Dorsal的保守氨基酸序列分别设计简并引物，从中国明对虾血细胞cDNA中先后克隆到了Relish和Dorsal基因的部分片段，并结合SMART-RACE技术分别获得了中国明对虾Relish基因（FcRelish）和Dorsal基因（FcDorsal）的cDNA 全长。 FcRelish的cDNA 全长为2157个碱基，其中开放阅读框为1512个碱基，编码504个氨基酸；FcRelish蛋白的推导分子量为57373.5 Da，理论等电点为7.00。FcDorsal基因的cDNA 全长为1627个碱基，其中开放阅读框为1071个碱基，编码357个氨基酸；FcDorsal蛋白的推导分子量为39780.7 Da，理论等电点为8.85。 分析了FcRelish基因和FcDorsal基因的在血细胞、淋巴器官、肠和肌肉等12个组织中的表达水平。组织表达结果表明FcRelish和FcDorsal在淋巴器官和血细胞中表达水平明显高于其他组织，而淋巴器官和血细胞是对虾免疫系统中最重要的两个组织，由此可以推测中国明对虾中的Relish和Dorsal可能与免疫关系密切。 本论文还利用实时荧光定量PCR技术，对灭活鳗弧菌刺激，以及WSSV病毒刺激后，对虾血细胞和淋巴器官中FcRelish基因和FcDorsal基因的转录水平进行了研究。FcRelish基因在WSSV病毒刺激后，在血细胞和淋巴器官中都出现了波浪形变化，说明FcRelish对WSSV病毒刺激产生了应答。在灭活鳗弧菌刺激后，FcRelish在血细胞中变化不明显，而在淋巴器官中出现了两次明显的下调上调交替，出现这种现象的具体原因有待探究。在WSSV病毒刺激后，血细胞和淋巴器官中FcDorsal的转录表达呈波浪形变化。而在鳗弧菌刺激后，FcDorsal在血细胞和淋巴器官中的转录均在短时间内出现明显的上调表达，说明FcDorsal对鳗弧菌非常敏感。 作为核转录因子的NF-κB蛋白的转录激活作用需要在细胞质中通过蛋白水解作用来激活，为了进一步阐明NF-κB对病菌感染的应答机制，需要进一步研究这两种转录因子在蛋白水平的变化，以便从分子水平阐明NF-κB在对虾天然免疫中的作用。