Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system^1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 μl during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds⁴. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish^5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies^1-3, congenital myopathies^4,5, and disruptions in sarcomeric assembly^6,7, due to high genomic and structural conservation with mammals⁸. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres⁹. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.The touch-evoked escape assay involves observing an embryo''s swimming behavior in response to tactile stimulation^10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance¹². The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.     

Functional Diversity of Glycinergic Commissural Inhibitory Neurons in Larval Zebrafish

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蛋白质组学在植物水分胁迫应答中的应用研究进展

水分胁迫是影响植物生长发育的主要生长因子。通过蛋白质组学技术可对水分胁迫下植物差异变化的蛋白和基因进行挖掘,在研究植物抗旱生理机制方面意义重大。总结了植物蛋白质组学的基本方法与关键技术,同时从光合与碳代谢相关蛋白、抗氧化系统、渗透调节蛋白、热激蛋白、胚胎发育晚期丰富蛋白、转录因子等方面综述了近几年国际上在植物水分胁迫蛋白质组研究方面的进展,并展望了今后蛋白质组学技术发展的方向。     

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

Whole-Brain Calcium Imaging during Physiological Vestibular Stimulation in Larval Zebrafish

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Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan

Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two‐dimensional gel electrophoresis and identified by matrix‐assisted laser desorption/ionization of time‐of‐flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co‐occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen‐related and energy‐production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies.     

Parallel Channels for Motion Feature Extraction in the Pretectum and Tectum of Larval Zebrafish

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Terminal Nerve GnRH3 Neurons Mediate Slow Avoidance of Carbon Dioxide in Larval Zebrafish

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不同抗性苹果品种应答轮纹病菌胁迫的差异蛋白质组分析

为鉴定不同抗性苹果(Malus domestica)品种响应轮纹病菌胁迫的抗性相关蛋白表达差异, 以抗病品种华月及易感品种金冠为试材, 采用高通量同位素标记定量(IBT)技术结合液相色谱-串联质谱(LC-MS)鉴定技术, 对病原菌处理前后抗、感病品种叶片的蛋白质组差异表达进行分析, 共鉴定出171个差异表达蛋白(DEPs)。GO富集及KEGG通路分析表明, 在细胞组分、分子功能和生物过程3类中共注释到686个GO条目, 其中52个DEPs注释于KEGG通路的18个显著差异途径(P<0.05)。亚细胞定位预测分析表明, 171个DEPs中有170个分别定位于8类细胞器。蛋白功能注释分析表明, 46个DEPs注释于7类抗性相关蛋白, 包括类甜蛋白、过氧化物酶、多酚氧化酶、过敏原蛋白、几丁质酶、内切葡聚糖酶以及主乳胶蛋白。此外, 还对抗性相关蛋白的表达特点及基因定量结果进行了分析。该研究结果可为进一步解析抗、感病苹果品种应答轮纹病菌胁迫的抗性机制提供参考。     

斑马鱼整体原位杂交的技术改良

斑马鱼整体原位杂交技术广泛应用于基因表达谱、基因间相互关系和突变体筛选等方面,是研究斑马鱼发育相关基因功能的重要技术。本文从杂交探针的制备、浓度的选择和洗脱以及胚胎的脱色、蛋白酶K消化、底物显色等方面进行了摸索、改进及简化,获得了背景低、着色清晰、特异性高的实验结果,预示了简单实用、成本低廉的斑马鱼整胚原位杂交技术平台的成功建立。     

Behavioral Plasticity in Response to Environmental Manipulation among Zebrafish (Danio rerio) Populations

Plastic responses can have adaptive significance for organisms occurring in unpredictable environments, migratory species and organisms occupying novel environments. Zebrafish (Danio rerio) occur in a wide range of habitats and environments that fluctuate frequently across seasons and habitats. We expect wild populations of fish to be behaviorally more flexible than fish reared in conventional laboratory and hatchery environments. We measured three behavioral traits among 2 wild (U and PN) and 1 laboratory bred (SH) zebrafish populations in four environments differing in water flow and vegetation regimes. We found that the degree of plasticity varied with the type of behavior and also among populations. In general, vegetation increased aggression and water flow decreased latency to feed after a disturbance, but the patterns were population dependent. For example, while wild U fish fed more readily after a disturbance in vegetated and/or flowing habitats, fish from the wild PN population and lab-reared SH strain showed little variation in foraging across different environmental conditions. Zebrafish from all the three populations were more aggressive when tested in an arena with vegetation. In contrast, while there was an inter- population difference in shoaling distances, variation in shoaling distance across environmental conditions within populations was not significant. These results suggest that both foraging and aggression in zebrafish are more plastic and influenced by immediate context than is shoaling distance, which may have a stronger genetic basis. Our findings point to different underlying mechanisms influencing the expression of these traits and warrants further investigations.     

原钙黏附蛋白18b基因抑制对斑马鱼神经系统发育的影响

原钙黏附蛋白18b(Protocadherin18b,Pcdh18b)属于钙黏附蛋白家族成员．为了研究pcdh18b基因抑制对斑马鱼神经系统发育的影响,针对pcdh18b的翻译起始位点设计一个吗啡啉修饰的反义寡核苷酸抑制其表达,在斑马鱼受精卵一到二细胞期注射并且验证其有效性．注射后用原位杂交和吖啶橙染色检测神经系统的表型和标志基因的表达．pcdh18b下调使神经前体细胞的标志基因neurog1、神经元标志基因elavl3和神经胶质细胞标志基因gfap的表达均出现下调,中后脑边界的标志基因pax2a和wnt1表达减弱并出现神经管分叉现象,同时与后脑分节相关的基因krox20表达减少．吖啶橙染色显示pcdh18b下调后斑马鱼中脑、后脑及中后脑边界细胞凋亡增多．这些结果表明pcdh18b抑制导致了斑马鱼神经系统发育的异常．     

Myocardial Polyploidization Creates a Barrier to Heart Regeneration in Zebrafish

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Proteome Changes Paralleling the Olfactory Conditioning in the Forager Honey Bee and Provision of a Brain Proteomics Dataset

The olfactory conditioning of the bee proboscis extension reflex (PER) is extensively used as a paradigm in associative learning of invertebrates but with limited molecular investigations. To investigate which protein changes are linked to olfactory conditioning, a non‐sophisticated conditioning model is applied using the PER in the honeybee (Apis mellifera). Foraging honeybees are assigned into three groups based on the reflex behavior and training: conditioned using 2‐octanone (PER‐conditioned), and sucrose and water controls. Thereafter, the brain synaptosomal proteins are isolated and analyzed by quantitative proteomics using stable isotope labeling (TMT). Additionally, the complex proteome dataset of the bee brain is generated with a total number of 5411 protein groups, including key players in neurotransmitter signaling. The most significant categories affected during olfactory conditioning are associated with “SNARE interactions in vesicular transport” (BET1 and VAMP7), ABC transporters, and fatty acid degradation pathways.