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A transgenic zebrafish model for in vivo long-term imaging of retinotectal synaptogenesis.

ABSTRACT: The retinotectal synapse in larval zebrafish, combined with live time-lapse imaging, provides an advantageous model for study of the development and remodelling of central synapses in vivo. In previous studies, these synapses were labelled by transient expression of fluorescence-tagged synaptic proteins, which resulted in the dramatic variation of labelling patterns in each larva. Here, using GAL4-Upstream Activating Sequence (GAL4-UAS) methodology, we generated stable transgenic lines, which express EGFP-tagged synaptophysin (a presynaptic protein) in retinal ganglion cells (RGCs), to reliably label the pre-synaptic site of retinotectal synapses. This tool avoids the variable labelling of RGCs that occurs in transient transgenic larvae. We obtained several stable transgenic lines that differ consistently in the number of labelled RGCs. Using stable lines that consistently had a single labelled RGC, we could trace synaptogenic dynamics on an individual RGC axonal arbor across different developmental stages. In the stable lines that consistently had multiple labelled RGCs, we could simultaneously monitor both pre- and post-synaptic compartments by combining transient labelling of post-synaptic sites on individual tectal neurons. These tools allowed us to investigate molecular events underlying synaptogenesis and found that the microRNA-132 (miR-132) is required for developmental synaptogenesis. Thus, these transgenic zebrafish stable lines provide appropriate tools for studying central synaptogenesis and underlying molecular mechanisms in intact vertebrate brain.

SUBMITTER: Du XF

PROVIDER: S-EPMC6145912 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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A transgenic zebrafish model for in vivo long-term imaging of retinotectal synaptogenesis.

Du Xu-Fei XF Xu Bing B Zhang Yu Y Chen Min-Jia MJ Du Jiu-Lin JL

Scientific reports 20180919 1

The retinotectal synapse in larval zebrafish, combined with live time-lapse imaging, provides an advantageous model for study of the development and remodelling of central synapses in vivo. In previous studies, these synapses were labelled by transient expression of fluorescence-tagged synaptic proteins, which resulted in the dramatic variation of labelling patterns in each larva. Here, using GAL4-Upstream Activating Sequence (GAL4-UAS) methodology, we generated stable transgenic lines, which ex ...[more]

PMID: 30232367

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Project description:Importance:Cannabis is the most commonly used illicit drug in the world. Cannabinoids have been shown to modulate immune responses; however, the association of cannabis with neuroimmune function has never been investigated in vivo in the human brain. Objective:To investigate neuroimmune activation or 18-kDa translocator protein (TSPO) levels in long-term cannabis users, and to evaluate the association of brain TSPO levels with behavioral measures and inflammatory blood biomarkers. Design, Setting, and Participants:This cross-sectional study based in Toronto, Ontario, recruited individuals from January 1, 2015, to October 30, 2018. Participants included long-term cannabis users (n?=?24) and non-cannabis-using controls (n?=?27). Cannabis users were included if they had a positive urine drug screen for only cannabis and if they used cannabis at least 4 times per week for the past 12 months and/or met the criteria for cannabis use disorder. All participants underwent a positron emission tomography scan with [18F]FEPPA, or fluorine F 18-labeled N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide. Main Outcomes and Measures:Total distribution volume was quantified across regions of interest. Stress and anxiety as well as peripheral measures of inflammatory cytokines and C-reactive protein levels were also measured. Results:In total, 24 long-term cannabis users (mean [SD] age, 23.1 [3.8] years; 15 men [63%]) and 27 non-cannabis-using controls (mean [SD] age, 23.6 [4.2] years; 18 women [67%]) were included and completed all study procedures. Compared with the controls, cannabis users had higher [18F]FEPPA total distribution volume (main group effect: F1,48?=?6.5 [P?=?.01]; ROI effect: F1,200?=?28.4 [P?<?.001]; Cohen d?=?0.6;?23.3% higher), with a more prominent implication for the cannabis use disorder subgroup (n?=?15; main group effect: F1,39?=?8.5 [P?=?.006]; ROI effect: F1,164?=?19.3 [P?<?.001]; Cohen d?=?0.8;?31.5% higher). Greater TSPO levels in the brain were associated with stress and anxiety and with higher circulating C-reactive protein levels in cannabis users. Conclusions and Relevance:The results of this study suggest that TSPO levels in cannabis users, particularly in those with cannabis use disorder, are higher than those in non-cannabis-using controls. The findings emphasize the need for more complementary preclinical systems for a better understanding of the role of cannabinoids and TSPO in neuroimmune signaling.

Project description:IntroductionThe gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to ?-ketoglutarate (?-KG) whereas mutant IDH1 converts ?-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants.Materials and methodsIDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage.ResultsIn total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity.ConclusionsWe have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation.

Dataset Information

A transgenic zebrafish model for in vivo long-term imaging of retinotectal synaptogenesis.

Publications

A transgenic zebrafish model for in vivo long-term imaging of retinotectal synaptogenesis.

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