NP394神经元控制果蝇趋避光行为

近日,Science杂志在线发表了中国科学院生物物理研究所刘力课题组龚哲峰副研究员等人关于发现果蝇幼虫中央脑的两对神经元足以调节果蝇幼虫对于不同光强条件的偏好行为的研究成果。     

果蝇幼虫的视觉系统

视觉对于动物的生存和行为来说是非常重要的。虽然果蝇幼虫的视觉神经系统在组织结构上比成虫简单,但是也具有一定的复杂性和多样性。在最近几年中,随着对果蝇幼虫视觉系统功能的研究取得重要进展,我们对于果蝇幼虫视觉系统的认识更加丰富了。果蝇幼虫视觉系统的结构中,最初级的光感受神经元包括三类,一类是BO/BN(Bolwig's organ/Bolwig's nerve),一类是表达感光分子CRY(cryptochrome)的神经元,另外一类是Ⅳ型DA(classⅣdendriticarborization)神经元;果蝇幼虫视觉系统的次级神经元主要是光节律相关的侧神经元(lateralneurons,LN),它表达Per(period)、Tim(timeless)及Pdf(pigment dispersion factor)等节律相关的蛋白分子;而第三级神经元包括更为下游的、表达果蝇促胸腺激素且直接调控幼虫光偏好的NP394神经元。这三级神经元构成了我们现在所了解的果蝇幼虫视觉神经系统的基本框架。     

让蚊子找不到要咬的人

很多昆虫具有能探测二氧化碳的神经元，而这种神经元的作用却因昆虫生态习性的不同而不同。比如说天蛾，它们用它来测量曼陀罗花的质量。而蚊子和其他食血昆虫则会被我们所发出的二氧化碳吸引。果蝇也有这些传感器，研究人员已经在其身上识别出了2种受体：Gr21a和Gr63a。只有其中一种受体的果蝇对二氧化碳不敏感。     

猫皮层SⅠ区神经元对刺激VPL的反应及其膜电学特性的研究

本文采用细胞内记录技术,研究了猫皮层第一躯体感觉区（ｐｒｉｍａｒｙｓｏｍａｔｏｓｅｎｓｏｒｙｃｏｒｔｅｘａｒｅａ,ＳⅠ区）躯体伤害感受神经元膜的电学特性和对刺激腹后外侧核（ｖｅｎｔｒａｌｐｏｓｔｅｒｉｏｒｌａｔｅｒａｌｎｕｃｌｅｕｓ,ＶＰＬ核）的反应。极化电流绝对值小于或等于１０ｎＡ时,伤害感受神经元ＩＶ极相关（ｒ＝０９６）,整流作用不明显;极化电流绝对值大于１０ｎＡ时,在两个方向上发生整流,ＩＶ曲线表现为Ｓ型,其中伤害感受神经元的整流作用较非伤害感受神经元明显。伤害性感受神经元Ｒｍ、τ、Ｃｍ明显大于非伤害感受神经元（Ｐ＜００１或Ｐ＜００５）。刺激ＶＰＬ与刺激隐神经在ＳⅠ区伤害感受神经元的诱发反应中存有相似与不同两种形式。用细胞内电位记录方法证明了单一神经元有会聚现象。结果提示,ＳⅠ区伤害感受神经元与非伤害感受神经元可能在细胞膜形态结构、细胞体积大小等方面存在有意义的差别,从而反映其不同的生理功能。     

皮层神经元动作电位不应期和阈电位与神经元动作电位编码的关系

目的：测量和比较感觉运动皮层Ⅱ/Ⅲ层锥体神经元和中间神经元的内在特性并研究其与动作电位编码频率和精确性的关系。方法：采用全细胞电流钳记录模式,获得的数据输入pClamp和Origin进行处理分析。结果：与锥体神经元相比,中间神经元群集动作电位具有较低的阈电位水平和较短的不应期,从而中间神经元具有较高的动作电位编码频率和精确性。结论：皮层神经元动作电位的阈电位水平和不应期调控动作电位的编码频率和精确性。     

两种果蝇成虫与幼虫期的竞争及其对二者共存的影响

昆虫均为完全或不完全变态发育,其幼虫和成虫阶段往往有着不同的资源需求。研究昆虫在幼虫和成虫阶段的生态位和适合度,有助于提升我们对昆虫物种共存和群落构建的认识。黑腹果蝇(Drosophilamelanogaster)和伊米果蝇(D.immigrans)是全球广布的两种果蝇,它们常常发生在相同的季节,且均在腐烂的水果上产卵,幼虫寄生在其中完成生长发育。本研究通过转瓶实验评估了这两种果蝇在连续竞争过程中的内禀增长率和种内与种间竞争系数,并进一步检验了它们的成虫对产卵场所,以及幼虫对食物的竞争强度,据此计算了两个物种在成虫和幼虫阶段的生态位分化与适合度差异,在当代物种共存理论的框架下分析了影响两种果蝇共存的关键因素。结果表明,连续饲养过程中,黑腹果蝇表现出更高的适合度,大概率会竞争排斥掉伊米果蝇。具体而言,两种果蝇在幼虫和成虫期均有极大的生态位重叠,虽然伊米果蝇成虫对产卵场所有着更高的利用率,黑腹果蝇的幼虫在生长发育阶段对饲料有着更高的利用率,但两种果蝇在成虫、幼虫阶段竞争的结果更多地取决于谁先占据资源。本研究表明昆虫在不同发育阶段对资源利用率的变化会在一定程度上影响它们共存的可能性。     

植物乳杆菌、苹果醋酸杆菌和酿酒酵母三种微生物对黑腹果蝇行为和发育的影响

【目的】肠道微生物在宿主的多种生命活动中发挥重要作用。本研究旨在通过研究植物乳杆菌Lactobacillus plantarum, 苹果醋酸杆菌Acetobacter malorum和酿酒酵母Saccharomyces cerevisiae 3种微生物对黑腹果蝇Drosophila melanogaster觅食、产卵和发育的影响,进一步阐明肠道微生物与宿主的互作调控机制。【方法】采用引诱实验检测体外培养的植物乳杆菌、苹果醋酸杆菌和酿酒酵母对交配前后黑腹果蝇成虫的引诱力以及这3种微生物的单一微生物和其中2种或3种微生物的混合物对非处女蝇的引诱力。采用产卵选择实验检测黑腹果蝇非处女蝇对单一微生物和其中2种或3种微生物的混合物的产卵偏好。将黑腹果蝇卵在接种分别含有植物乳杆菌、苹果醋酸杆菌和酿酒酵母的培养基和正常培养基(对照)上培养,测量幼虫体重;同时将卵在分别含有活菌或灭活的这3种单一微生物的培养基上和正常培养基上培养,统计化蛹数,以检测不同微生物对黑腹果蝇幼虫发育的影响。将黑腹果蝇幼虫在分别含有植物乳杆菌、苹果醋酸杆菌和酿酒酵母的培养基及正常培养基中培养72 h,qRT-PCR检测胰岛素信号通路的关键基因InR在黑腹果蝇幼虫中的相对表达量变化。【结果】苹果醋酸杆菌主要影响黑腹果蝇的产卵,酿酒酵母和植物乳杆菌则可同时影响黑腹果蝇觅食和产卵,且与单一微生物相比,非处女黑腹果蝇更趋向于含有2种或3种微生物的混合物。产卵选择实验表明,3种单一供试微生物及其中2种或3种微生物的混合物对黑腹果蝇的产卵选择具有显著的吸引力,其中混合物的影响最大,然后依次是苹果醋酸杆菌、酿酒酵母、植物乳杆菌。3种微生物均可促进黑腹果蝇幼虫的发育,活菌在接种早期加快幼虫到蛹的发育,灭活菌的促进作用则相对滞后。与对照比较,InR在接种苹果醋酸杆菌的培养基上培养的黑腹果蝇幼虫中的表达量显著降低,而在分别接种植物乳杆菌和酿酒酵母的培养基上培养的幼虫中则被显著促进表达。【结论】苹果醋酸杆菌可引诱黑腹果蝇的产卵,酿酒酵母和植物乳杆菌对黑腹果蝇的觅食和产卵都有具有引诱力;同时微生物的多样性可增加黑腹果蝇的觅食和产卵偏好。植物乳杆菌、苹果醋酸杆菌和酿酒酵母可能通过不同的调控机制影响黑腹果蝇幼虫的生长发育。     

SKF-96365对SD大鼠急性分离下丘脑神经元温度敏感性的影响

采用电流钳技术观察SD大鼠急性分离下丘脑神经元的自发性放电及其温度敏感特性,显微镜下,急性分离神经元表现为两种形态,一种具有突起,另一种表现为无突起的形态,记录到的有突起的78例神经元中,可以记录到4例热敏神经元（wsn）,但在无突起的61例神经元没有观察到热敏神经元的存在．在浴槽液体中加入SKF96365,这些热敏神经元就会失去温度敏感性．以上结果表明pWSN可能通过突起部位上分布的TRPV4而起到对周围环境温度的感受作用．     

蜜蜂下行神经元的光谱特性

利用生理细胞外记录方法研究了蜜蜂下行神经元的光谱敏感特性,不同波长的单色光刺激引起的给先反应和撤光反应的相对强度不同．分析了３７例下行神经元的光谱敏感特性,发现下行神经元有的是光谱宽带和多光谱性的,有的是光谱窄带,给光和撤光反应都具有不同的最敏感区。     

电突触耦合Chay神经元同步振荡的研究

从微观解释异常神经元构建组织时癫痫样波形的相互制约关系对神经系统疾病的研究很有意义,而两神经元耦合特性的探索是重要的基础工作。采用Chay提供的Pacemaker神经元模型以电突触耦合来研究不同耦合强度对神经元动态时序的影响,并指出突触作用过程的混沌特征。给出并讨论了不同状态神经元相耦合时非线性振荡的数值计算结果,即：起搏神经元与处于冲动混沌状态神经元、处于冲动混沌和独态冲动状态的异常神经元、异常神经元与处于静息状态神经元的动态时序,还给出了部分相图以及Ca 离子浓度变化的特点。神经元这种负载特性的讨论有助于研究在活组织中癫痫发作的机理、传输和控制。     

Altered outward K(+) currents in Drosophila larval neurons of memory mutants rutabaga and amnesiac.

K(+) currents in cultured Drosophila larval neurons have been classified into four categories according to their inactivation time constants, relative amplitude, and response to K(+) channel blockers 4-AP and tetraethylammonium. The percentage (65%) of neurons displaying K(+) currents which were reduced to 30% in amplitude by 5 mM cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP in both Drosophila memory mutants rutabaga (rut) and amnesiac (amn) was significantly larger than that (50%) in wild type. This initial characterization provides evidence for altered K(+) currents in both rut and amn mutants. Arachidonic acid, a specifical inhibitor of Kv4 family (shal) K(+) channels, was found to inhibit K(+) currents in cultured Drosophila neurons, suggesting the presence of shal channels in these neurons.     

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholesterol-depleting compounds modulate K+-currents in Drosophila Kenyon cells

Sterol-enriched lipid rafts have been involved in Drosophila membrane signalling such as Hedgehog targeting and glutamate receptor ligand-affinity regulation. Here, we show that the voltage-dependent K(+) currents expressed by the intrinsic neurons of the Mushroom bodies are upward-modulated by compounds that remove sterols from the plasma membrane. Modulation seems to rely on a fast-exchanging sterol-pool, which more strongly affects the slowly inactivating current. Our results provide the first evidence that sterols influence the operation of voltage-gated ion channels in Drosophila neurons and strengthen the importance of lipid rafts in this biological model.     

Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons

The major component of whole-cell Ca2+ current in differentiated, neuron-like rat pheochromocytoma (PC12) cells and sympathetic neurons is carried by dihydropyridine-insensitive, high-threshold-activated N-type Ca2+ channels. We show that these channels have unitary properties distinct from those of previously described Ca2+ channels and contribute both slowly inactivating and large sustained components of whole-cell current. The N-type Ca2+ currents are modulated by GTP binding proteins. The snail toxin omega-conotoxin reveals two pharmacological components of N-type currents, one blocked irreversibly and one inhibited reversibly. Contrary to previous reports, neuronal L-type channels are insensitive to omega-conotoxin. N-type Ca2+ channels appear to be specific for neuronal cells, since their functional expression is greatly enhanced by nerve growth factor.     

Characterization of voltage-and Ca2+-activated K+ channels in rat dorsal root ganglion neurons

Auxiliary beta-subunits associated with pore-forming Slo1 alpha-subunits play an essential role in regulating functional properties of large-conductance, voltage- and Ca(2+)-activated K(+) channels commonly termed BK channels. Even though both noninactivating and inactivating BK channels are thought to be regulated by beta-subunits (beta1, beta2, beta3, or beta4), the molecular determinants underlying inactivating BK channels in native cells have not been extensively demonstrated. In this study, rbeta2 (but not rbeta3-subunit) was identified as a molecular component in rat lumbar L4-6 dorsal root ganglia (DRG) by RT-PCR responsible for inactivating large-conductance Ca(2+)-dependent K(+) currents (BK(i) currents) in small sensory neurons. The properties of native BK(i) currents obtained from both whole-cell and inside-out patches are very similar to inactivating BK channels produced by co-expressing mSlo1 alpha- and hbeta2-subunits in Xenopus oocytes. Intracellular application of 0.5 mg/ml trypsin removes inactivation of BK(i) channels, and the specific blockers of BK channels, charybdotoxin (ChTX) and iberiotoxin (IbTX), inhibit these BK(i) currents. Single BK(i) channel currents derived from inside-out patches revealed that one BK(i) channel contained three rbeta2-subunits (on average), with a single-channel conductance about 217 pS under 160 K(+) symmetrical recording conditions. Blockade of BK(i) channels by 100 nM IbTX augmented firing frequency, broadened action potential waveform and reduced after-hyperpolarization. We propose that the BK(i) channels in small diameter DRG sensory neurons might play an important role in regulating nociceptive input to the central nervous system (CNS).     

Modification of K channel inactivation by papain and N-bromoacetamide.

The whole-cell variation of the patch clamp technique was used to study macroscopic K current in voltage clamped GH3 cells. An inactivating, voltage-dependent K current was studied in isolation by inhibiting Ca-activated K currents with internal Ca chelators and external tetraethylammonium ions. Under control conditions, the K current inactivated in two phases with time constants of 25 and 79 ms. After treatment with either a proteolytic enzyme such as papain or the amino acid reagent N-bromoacetamide, the K current no longer inactivated rapidly, but decayed very slowly with a time constant of 500 to 750 ms. The action of papain or N-bromoacetamide on K channels is comparable to their action on Na channels, suggesting that inactivation in Na and K channels occurs by a similar mechanism.     

Level of expression controls modes of gating of a K+ channel.

Several distinct subfamilies of K+ channel genes have been discovered by molecular cloning, however, in some cases the structural differences among them do not account for the diversity of K+ current types, ranging from transient A-type to slowly inactivating delayed rectifier-type, as members within each subfamily have been shown to code for K+ channels of different inactivation kinetics and pharmacological properties. We show that a single K+ channel cDNA of the Shaker subfamily (ShH4) can express in Xenopus oocytes not only a transient A-type K+ current but also, upon increased level of expression, slowly inactivating K+ currents with markedly reduced sensitivity to tetraethylammonium. In correlation with the macroscopic currents there are single-channel gating modes ranging from the fast-inactivation mode which underlies the transient A-type current, to slow-inactivation modes characterized by bursts of longer openings, and corresponding to the slowly inactivating macroscopic currents.     

The Drosophila Shaker gene codes for a distinctive K+ current in a subset of neurons

A transient K+ current coded by the Shaker gene was identified in muscle and expressed in Xenopus oocytes by injecting cRNA transcribed from a cloned cDNA. The Shaker current has not previously been identified in neurons. Mutational analysis now reveals that in neurons, Shaker is required for expression of a very rapidly inactivating K+ current with a depolarized steady-state inactivation curve. Together, these properties distinguish the Shaker-coded current from similar fast transient K+ currents coded by other genes. The Sh5 mutation further enhanced the depolarization of the Shaker current steady-state inactivation curve. Deletion of the Shaker gene completely removes the transient K+ current from a small percentage of neurons (15%) in a mixed population, and removes a portion of the whole-cell current in about 35% of neurons. The remaining 50% of neurons were apparently unaffected by deletion of the Shaker gene. The unique combination of rapid inactivation and depolarized steady-state inactivation of the Shaker current may reflect a unique functional role for this current in the nervous system such as the rapid repolarization of action potentials.     

Estradiol feedback alters potassium currents and firing properties of gonadotropin-releasing hormone neurons

GnRH neurons are regulated by estradiol feedback through unknown mechanisms. Voltage-gated potassium channels determine the pattern of activity and response to synaptic inputs in many neurons. We used whole-cell patch-clamp to test whether estradiol feedback altered potassium currents in GnRH neurons. Adult mice were ovariectomized and some treated with estradiol implants to suppress reproductive neuroendocrine function; 1 wk later, brain slices were prepared for recording. Estradiol affected the amplitude, decay time, and the voltage dependence of both inactivation and activation of A-type potassium currents in these cells. Estradiol also altered a slowly inactivating current, I(K.) The estradiol-induced changes in I(A) contributed to marked changes in action potential properties. Estradiol increased excitability in GnRH neurons, decreasing both threshold and latency for action potential generation. To test whether estradiol altered phosphorylation of the channels or associated proteins, the broad-spectrum kinase inhibitor H7 was included in the recording pipette. H7 acutely reversed some but not all effects of estradiol on potassium currents. Estradiol did not affect I(A) or I(K) in paraventricular neurosecretory neurons, demonstrating a degree of specificity in these effects. Potassium channels are thus one target for estradiol regulation of GnRH neurons; this regulation involves changes in phosphorylation of potassium channel components.     

K(+) currents responsible for repolarization in mouse ventricle and their modulation by FK-506 and rapamycin

Modulation of mouse ventricular action potentials and K(+) currents was examined using the whole cell patch-clamp technique. The composite mouse ventricular K(+) current (consisted of an outward transient followed by a slowly decaying sustained component. Use of the K(+) channel blockers tetraethylammonium and 4-aminopyridine and a transgenic mouse model revealed three pharmacologically and kinetically distinct currents: I(to), which contributed to the transient component; I(K), which contributed to the sustained component; and a slowly activating current (I(slow)), which contributed to both components. The immunosuppressant FK-506 increased action potential duration at 90% repolarization by 66.7% by decreasing the sustained component (-48% at +60 mV) and prolonging recovery from inactivation (by 26% at 200 ms) of the transient component. These effects were isolated to I(K) and I(to), respectively. Rapamycin had strikingly similar effects on these currents. Both FK-506 and rapamycin are known to target the immunophilin FKBP12. Thus we conclude that FKBP12 modulates specific mouse K(+) channels, and thus the mouse ventricular action potential, by interacting directly with K(+) channel proteins or with other associated regulatory proteins.