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。烟碱受体在神经系统中广泛发现，并在行为和病理状况的各个方面起重要作用。具体而言，它们的参与与酒精中毒密切相关。我们使用斑马鱼的神经系统作为实验模型。在这个财政年度，由我的部分中进行的基因筛查和两篇研究神经系统生理学的论文产生的两篇论文。 斑马鱼罗汉 - 波尔德神经元中电压门控Ca2+电流的题为鉴定和调节的手稿发表在《神经生理学杂志》上。摘要如下。电兴奋的细胞在质膜上具有依赖电压依赖的离子通道，可调节膜渗透性对特定离子。电压门控Ca2+通道（VGCC）尤为重要，因为CA2+既用充电载体和第二使者。斑马鱼（Danio Rerio）是研究神经元兴奋性，电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尚未探索，因为缺乏适合全细胞电压钳研究的准备。 Rohon-Beard（R-B）感觉神经元代表了此目的的有吸引力的候选者，因为它们相对较大，并且与哺乳动物背根神经元（DRG）神经元具有功能性同源性。 R-B神经元（ISL2B：EGFP）ZC7中表达绿色荧光蛋白的转基因斑马鱼被用于鉴定适合全细胞贴剂钳实验的解离神经元。基于生物物理和药理特性，斑马鱼R-B神经元表达高压和低压门控的Ca2+电流（分别为HVA-和LVA-KIA）。 Ni+ - 敏感的LVA-KIA发生在少数R-B神经元（30％）和对GVIA敏感的CAV2.2（N型）Ca2+通道中，这是HVA-MICA的绝大多数（90％）。为了鉴定调节HVA-KIA的G蛋白偶联受体（GPCR），筛选了一组神经递质。 GABA/BACLOFEN或5-羟色胺的应用产生了依赖电压的抑制作用，而使用Mu-Apoil agonist Damgo则导致了无效的抑制作用。与哺乳动物神经元不同，GPCR介导的ICA的电压依赖性调节似乎主要是通过霍乱毒素敏感的Gα亚基转导的。这些结果为使用斑马鱼模型系统来理解Ca2+通道函数提供了基础，而Ca2+通道如何对机械感应函数有贡献。 斑马鱼中出版了一种题为鉴定成年肾脏祖细胞和能够再生的成年肾脏祖细胞。弃权如下。肾功能的丧失是许多肾脏疾病的基础。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，Fish在整个生命周期中添加了肾单位，并在受伤后再生肾脏再生，这提供了一种模型，以了解如何治疗哺乳动物肾脏再生。在这里，我们将成年斑马鱼中新肾脏的来源追踪到包含肾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年人并产生多个肾单位。测试自我更新的序列移植实验表明，肾祖细胞长期存在，具有显着的复制潜力，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镶嵌肾脏，表明多个肾单位祖细胞有助于单个肾单位。与此相一致，透明幼虫中肾单位形成的活成像表明，肾脏骨料通过多个细胞的合并形成，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包含自我更新的肾单茎/祖细胞。这些细胞的鉴定铺平了在哺乳动物中分离或设计等效细胞并开发新型肾脏再生疗法的方法。 由斑马鱼特异性PAX基因确定的斑马鱼中脊柱网络形成的手稿发表在《比较神经病学杂志》上。摘要如下。在脊柱网络的形成中，各种转录因子相互作用以开发特定的细胞类型。通过使用基因陷阱技术，我们建立了一条稳定的斑马鱼线，其中将红色荧光蛋白（RFP）插入PAX8基因中。 RFP插入标记了荧光的推定PAX8-LINEGE细胞，并抑制了纯合胚胎中的PAX8表达。 PAX8纯合胚胎在耳囊泡中显示出缺陷，如形态学研究中先前报道。与过早死亡的哺乳动物对应物相比，PAX8纯合胚胎成年了。 RFP在背脊髓中表达。对轴突形态的检查表明，RFP+神经元包括合理的分叉纵向（COBL）中间神经元，但其他抑制性神经元（例如连接局部（COLO）中间神经元和周环上升（CIA）中internerons interneurons insneurons s note dembiondery神经元都没有表达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纯合鱼中，RFP+细胞的分化与PAX8杂合鱼相似，而游泳行为仍然完好无损。相反，PAX2A/PAX8双突变体的RFP+细胞显示出改变的细胞命运。未观察到cobl。取而代之的是，RFP+细胞表现出轴突的同侧，形态类似于V2A/V2B中间神经元的形态。 一项题为“乙酰胆碱受体缺乏伽玛和epsilon”亚基的手稿介导了斑马鱼慢肌突触中的传播。弃权如下。幼虫斑马鱼中的快速和缓慢的骨骼肌类型可以通过其突触衰减的时间过程差异五倍。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）同工型赋予的。通过克隆斑马鱼Achr亚基cDNA并在Xenopus laevis卵母细胞中表达这种区别的基本基础。单通道电导和平均开放式爆发持续时间分配的alpha2; beta; delta; epsilon到快速肌肉突触电流的测量值。与期望相反，仅由alpha; beta; delta亚基（假定为alpha2; beta; delta2受体）组成的受体概括了缓慢肌肉单通道电流的动力学和电导。支持γ/epsilon的受体作为缓慢肌肉突触的介体的其他证据反映在异源表达的α2; beta; delta2受体的内向整流中，beta2受体是一种通常与神经元型烟碱型烟碱型受体相关的特性。在慢肌中，单通道和突触电流都反映了类似的矫正，从而将它们与快速肌肉区分开。缓慢肌肉中α2; beta; delta2受体的最终证据是通过我们将快速肌肉突触电流转化为慢肌的能力，通过击倒体内Epsilon Subunit的表达。因此，首次可以将肌肉突触功能归因于仅由三个不同亚基组成的受体同工型。 alpha2; beta; delta2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方面起着核心作用。