In vivo studies of neural system in zebrafish

斑马鱼神经系统的体内研究

• 批准号: 8344691
• 负责人: Fumihito Ono
• 金额: $ 82.08万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344691
• 关键词: Adult; Afferent Neurons; Agonist; Alcoholism; Axon; Baclofen; Behavior; Biological Models; Cell Lineage; Cell Membrane Permeability; Cell membrane; Cells; Characteristics; Charge; Cholera Toxin; Cholinergic Receptors; Cloning; Complementary DNA; Conotoxin; Data; Defect; Development; Dorsal; Embryo; Engineering; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Exhibits; Experimental Models; Fishes; Fluorescence; G-Protein-Coupled Receptors; Genes; Genetic; Genetic Screening; Green Fluorescent Proteins; Image; Injury; Interneurons; Ion Channel; Ions; Journals; Kidney; Kidney Diseases; Kinetics; Larva; Life; Longevity; Mammals; Manuscripts; Measurement; Mediating; Mediator of activation protein; Minority; Modeling; Morphology; Muscle; Natural regeneration; Nature; Nephrons; Nervous System Physiology; Nervous system structure; Neurology; Neurons; Neurotransmitters; Nicotinic Receptors; Oocytes; Opioid; Optics; Otic Vesicle; Paper; Physiology; Play; Preparation; Property; Protein Isoforms; Publishing; Renal function; Reporting; Role; Second Messenger Systems; Serotonin; Skeletal Muscle; Source; Spinal; Spinal Cord; Spinal Ganglia; Stem cells; Swimming; Synapses; System; Techniques; Testing; Time; Transgenic Organisms; Transplantation; Vertebrates; Xenopus laevis; Zebrafish; abstracting; alcohol response; base; cell type; comparative; expectation; gamma-Aminobutyric Acid; in vivo; inhibitory neuron; knock-down; mutant; neuronal cell body; neuronal excitability; neurophysiology; novel; patch clamp; progenitor; receptor; red fluorescent protein; regenerative therapy; relating to nervous system; repaired; research study; second messenger; self-renewal; stem; synaptic function; synaptogenesis; transcription factor; transmission process; voltage; voltage clamp

Projects in my section aim at clarifying the mechanisms of synapse formation and function, with an emphasis on the nicotinic synapses. Nicotinic receptors are found widely in the nervous system, and play important roles in various aspects of behavior and pathological conditions. Specifically, their involvement has been strongly implicated in alcoholism. We use the nervous system of zebrafish as the experimental model. In this fiscal year, two papers resulting from a genetic screening conducted in my section and two papers examining the physiology of nervous system were published. A manuscript entitled Identification and modulation of voltage-gated Ca2+ current in zebrafish Rohon-Beard neurons was published in the Journal of Neurophysiology. The abstract is as follows. Electrically excitable cells have voltage-dependent ion channels on the plasma membrane that regulate membrane permeability to specific ions. Voltage-gated Ca2+ channels (VGCCs) are especially important as Ca2+ serves as both a charge carrier and second messenger. Zebrafish (Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, electrophysiological properties of zebrafish VGCCs remain largely unexplored because a suitable preparation for whole cell voltage-clamp studies is lacking. Rohon-Beard (R-B) sensory neurons represent an attractive candidate for this purpose because of their relatively large somata and functional homology to mammalian dorsal root ganglia (DRG) neurons. Transgenic zebrafish expressing green fluorescent protein in R-B neurons, (Isl2b:EGFP)ZC7, were used to identify dissociated neurons suitable for whole cell patch-clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-gated Ca2+ current (HVA- and LVA-ICa, respectively). Ni+-sensitive LVA-ICa occur in the minority of R-B neurons (30%) and ω-conotoxin GVIA-sensitive CaV2.2 (N-type) Ca2+ channels underlie the vast majority (90%) of HVA-ICa. To identify G protein coupled receptors (GPCRs) that modulate HVA-ICa, a panel of neurotransmitters was screened. Application of GABA/baclofen or serotonin produced a voltage-dependent inhibition while application of the mu-opioid agonist DAMGO resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of ICa appears to be transduced primarily via a cholera toxin-sensitive Gα subunit. These results provide the basis for using the zebrafish model system to understanding Ca2+ channel function, and in turn, how Ca2+ channels contribute to mechanosensory function. A manuscript entitled Identification of adult renal progenitor cells capable of nephron formation and regeneration in zebrafish was published in Nature. The abstact is as follows. Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10-30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies. A manuscript entitled Formation of the spinal network in zebrafish determined by domain-specific Pax genes was published in the Journal of Comparative Neurology. The abstract is as follows. In the formation of the spinal network, various transcription factors interact to develop specific cell types. By using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted into the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies with morpholinos. The pax8 homozygous embryos survived to adulthood, in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP+ neurons include commissural bifurcating longitudinal (CoBL) interneurons, but other inhibitory neurons such as commissural local (CoLo) interneurons and circumferential ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP+ cells underwent differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP+ cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP+ cells exhibited axons descending ipsilaterally, a morphology resembling that of V2a/V2b interneurons. A manuscript entitled An acetylcholine receptor lacking both gamma and epsilon subunits mediates transmission in zebrafish slow muscle synapses was published in the Journal of General Physiology. The abstact is as follows. Fast and slow skeletal muscle types in larval zebrafish can be distinguished by a fivefold difference in the time course of their synaptic decay. Single-channel recordings indicate that this difference is conferred through kinetically distinct nicotinic acetylcholine receptor (AChR) isoforms. The underlying basis for this distinction was explored by cloning zebrafish muscle AChR subunit cDNAs and expressing them in Xenopus laevis oocytes. Measurements of single-channel conductance and mean open burst duration assigned alpha2;beta;delta;epsilon to fast muscle synaptic current. Contrary to expectations, receptors composed of only alpha;beta;delta subunits (presumed to be alpha2;beta;delta2 receptors) recapitulated the kinetics and conductance of slow muscle single-channel currents. Additional evidence in support of gamma/epsilon-less receptors as mediators of slow muscle synapses was reflected in the inward current rectification of heterologously expressed alpha2;beta;delta2 receptors, a property normally associated with neuronal-type nicotinic receptors. Similar rectification was reflected in both single-channel and synaptic currents in slow muscle, distinguishing them from fast muscle. The final evidence for alpha2;beta;delta2 receptors in slow muscle was provided by our ability to convert fast muscle synaptic currents to those of slow muscle by knocking down epsilon subunit expression in vivo. Thus, for the first time, muscle synaptic function can be ascribed to a receptor isoform that is composed of only three different subunits. The unique functional features offered by the alpha2;beta;delta2 receptor likely play a central role in mediating the persistent contractions characteristic to this muscle type.

我的部分的项目旨在阐明突触形成和功能的机制，重点是烟碱突触。烟碱受体广泛存在于神经系统中，并在行为和病理状况的各个方面发挥重要作用。具体来说，他们的参与与酗酒密切相关。我们使用斑马鱼的神经系统作为实验模型。在本财年，我的部门发表了两篇基因筛查论文和两篇研究神经系统生理学的论文。 题为“斑马鱼 Rohon-Beard 神经元中电压门控 Ca2+ 电流的识别和调制”的手稿发表在《神经生理学杂志》上。摘要如下。电兴奋细胞的质膜上具有电压依赖性离子通道，可调节膜对特定离子的通透性。电压门控 Ca2+ 通道 (VGCC) 尤其重要，因为 Ca2+ 既充当电荷载体又充当第二信使。斑马鱼 (Danio rerio) 是研究神经元兴奋性、回路和行为的重要模型脊椎动物。然而，斑马鱼 VGCC 的电生理特性在很大程度上仍未得到探索，因为缺乏适合全细胞电压钳研究的制剂。 Rohon-Beard (R-B) 感觉神经元是这一目的的一个有吸引力的候选者，因为它们相对较大的体细胞和与哺乳动物背根神经节 (DRG) 神经元的功能同源性。在 R-B 神经元中表达绿色荧光蛋白的转基因斑马鱼 (Isl2b:EGFP)ZC7 用于鉴定适合全细胞膜片钳实验的分离神经元。根据生物物理和药理学特性，斑马鱼 R-B 神经元表达高电压和低电压门控 Ca2+ 电流（分别为 HVA- 和 LVA-ICa）。 Ni+ 敏感的 LVA-ICa 出现在少数 R-B 神经元 (30%) 中，而 ω-芋螺毒素 GVIA 敏感的 CaV2.2（N 型）Ca2+ 通道是绝大多数 (90%) HVA-ICa 的基础。为了鉴定调节 HVA-ICa 的 G 蛋白偶联受体 (GPCR)，筛选了一组神经递质。应用 GABA/巴氯芬或血清素产生电压依赖性抑制，而应用 mu-阿片类激动剂 DAMGO 则产生电压非依赖性抑制。与哺乳动物神经元不同，GPCR 介导的 ICa 电压依赖性调节似乎主要通过霍乱毒素敏感的 Gα 亚基进行转导。这些结果为使用斑马鱼模型系统了解 Ca2+ 通道功能以及 Ca2+ 通道如何促进机械感觉功能提供了基础。 题为《斑马鱼肾单位形成和再生能力的成体肾祖细胞的鉴定》的手稿发表在《自然》杂志上。摘要如下。肾功能丧失是许多肾脏疾病的根源。哺乳动物可以部分修复其肾单位（肾脏的功能单位），但不能形成新的肾单位。相比之下，鱼类在其整个生命周期中都会增加肾单位，并在受伤后重新再生肾单位，这为了解如何通过治疗激活哺乳动物肾再生提供了模型。在这里，我们追踪成年斑马鱼中新肾单位的来源，即含有肾单位祖细胞的小细胞聚集体。包含 10-30 个细胞的单个聚集体的移植足以植入成体并产生多个肾单位。测试自我更新的连续移植实验表明，肾单位祖细胞寿命较长，并具有显着的复制潜力，这与干细胞活性一致。用绿色或红色荧光蛋白标记的混合肾单位祖细胞的移植产生了一些嵌合肾单位，表明多个肾单位祖细胞有助于单个肾单位。与此一致的是，透明幼虫中肾单位形成的实时成像表明，肾源性聚集体是由多个细胞合并形成的，然后分化为肾单位。综上所述，这些数据表明斑马鱼肾脏可能含有自我更新的肾单位干/祖细胞。这些细胞的鉴定为在哺乳动物中分离或改造等效细胞以及开发新型肾脏再生疗法铺平了道路。 一篇题为“斑马鱼脊柱网络的形成由特定领域的 Pax 基因决定”的手稿发表在《比较神经学杂志》上。摘要如下。在脊髓网络的形成过程中，各种转录因子相互作用以形成特定的细胞类型。通过使用基因捕获技术，我们建立了一个稳定的斑马鱼品系，其中红色荧光蛋白（RFP）被插入到pax8基因中。 RFP 插入用荧光标记假定的 pax8 谱系细胞，并抑制纯合胚胎中的 pax8 表达。 Pax8 纯合胚胎在耳囊中表现出缺陷，正如先前在吗啉代研究中报道的那样。 pax8纯合胚胎能够存活到成年，而哺乳动物胚胎则过早死亡。 RFP 在背侧脊髓中表达。轴突形态检查显示，RFP+ 神经元包括连合分叉纵向 (CoBL) 中间神经元，但其他抑制性神经元，如连合局部 (CoLo) 中间神经元和环周上升 (CiA) 中间神经元不表达 RFP。我们检查了抑制 pax2a/pax8 表达对中间神经元发育的影响。在pax8纯合子鱼中，RFP+细胞经历了与pax8杂合子鱼类似的分化，并且游泳行为保持完整。相比之下，pax2a/pax8 双突变体的 RFP+ 细胞表现出改变的细胞命运。没有观察到 CoBL。相反，RFP+ 细胞表现出轴突同侧下降，其形态类似于 V2a/V2b 中间神经元。 一篇题为《缺乏伽玛和ε亚基的乙酰胆碱受体介导斑马鱼慢肌突触传递》的手稿发表在《普通生理学杂志》上。摘要如下。斑马鱼幼体的快速和慢速骨骼肌类型可以通过突触衰减时间过程的五倍差异来区分。单通道记录表明，这种差异是通过动力学上不同的烟碱乙酰胆碱受体（AChR）亚型赋予的。通过克隆斑马鱼肌肉 AChR 亚基 cDNA 并在非洲爪蟾卵母细胞中表达它们，探索了这种区别的根本基础。单通道电导和平均开放突发持续时间的测量分配给快速肌肉突触电流 α2;β;δ;ε。与预期相反，仅由α；β；δ亚基组成的受体（推测为α2；β；δ2受体）概括了慢肌单通道电流的动力学和电导。支持 γ/ε-less 受体作为慢肌突触调节剂的其他证据反映在异源表达的 α2;β;δ2 受体的内向电流校正中，该特性通常与神经元型烟碱受体相关。类似的整流也反映在慢肌的单通道电流和突触电流中，这将它们与快肌区分开来。我们通过降低体内ε亚基表达将快肌突触电流转换为慢肌突触电流，从而提供了慢肌中存在α2；β；δ2受体的最终证据。因此，肌肉突触功能第一次可以归因于仅由三个不同亚基组成的受体亚型。 α2；β；δ2 受体提供的独特功能特征可能在介导这种肌肉类型的持续收缩特征中发挥核心作用。