Recording the highly diverse and dynamic activities in large populations of neurons in behaving animals is crucial for a better understanding of how the brain works. To meet this challenge, extensive efforts have been devoted to developing functional fluorescent indicators and optical imaging techniques to optically monitor neural activity. Indeed, optical imaging potentially has extremely high throughput due to its non-invasive access to large brain regions and capability to sample neurons at high density, but the readout speed, such as the scanning speed in two-photon scanning microscopy, is often limited by various practical considerations. Among different imaging methods, light field microscopy features a highly parallelized 3D fluorescence imaging scheme and therefore promises a novel and faster strategy for functional imaging of neural activity. Here, we briefly review the working principles of various types of light field microscopes and their recent developments and applications in neuroscience studies. We also discuss strategies and considerations of optimizing light field microscopy for different experimental purposes, with illustrative examples in imaging zebrafish and mouse brains.
Animals ; Mice ; Microscopy/methods* ; Zebrafish ; Neurons/physiology* ; Brain/physiology* ; Neurosciences

Evading or escaping from predators is one of the most crucial issues for survival across the animal kingdom. The timely detection of predators and the initiation of appropriate fight-or-flight responses are innate capabilities of the nervous system. Here we review recent progress in our understanding of innate visually-triggered defensive behaviors and the underlying neural circuit mechanisms, and a comparison among vinegar flies, zebrafish, and mice is included. This overview covers the anatomical and functional aspects of the neural circuits involved in this process, including visual threat processing and identification, the selection of appropriate behavioral responses, and the initiation of these innate defensive behaviors. The emphasis of this review is on the early stages of this pathway, namely, threat identification from complex visual inputs and how behavioral choices are influenced by differences in visual threats. We also briefly cover how the innate defensive response is processed centrally. Based on these summaries, we discuss coding strategies for visual threats and propose a common prototypical pathway for rapid innate defensive responses.
Mice ; Animals ; Zebrafish ; Neurons/physiology* ; Visual Perception/physiology*

Predatory hunting is an important type of innate behavior evolutionarily conserved across the animal kingdom. It is typically composed of a set of sequential actions, including prey search, pursuit, attack, and consumption. This behavior is subject to control by the nervous system. Early studies used toads as a model to probe the neuroethology of hunting, which led to the proposal of a sensory-triggered release mechanism for hunting actions. More recent studies have used genetically-trackable zebrafish and rodents and have made breakthrough discoveries in the neuroethology and neurocircuits underlying this behavior. Here, we review the sophisticated neurocircuitry involved in hunting and summarize the detailed mechanism for the circuitry to encode various aspects of hunting neuroethology, including sensory processing, sensorimotor transformation, motivation, and sequential encoding of hunting actions. We also discuss the overlapping brain circuits for hunting and feeding and point out the limitations of current studies. We propose that hunting is an ideal behavioral paradigm in which to study the neuroethology of motivated behaviors, which may shed new light on epidemic disorders, including binge-eating, obesity, and obsessive-compulsive disorders.
Animals ; Zebrafish ; Hunting ; Predatory Behavior/physiology* ; Neurons/physiology* ; Motivation

BACKGROUNDFolic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2) and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations.

METHODSMorpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression.

RESULTSDHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P < 0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos.

CONCLUSIONSDHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; physiology ; Female ; Heart ; embryology ; Heart Defects, Congenital ; etiology ; Tetrahydrofolate Dehydrogenase ; genetics ; physiology ; Zebrafish ; Zebrafish Proteins ; genetics ; physiology

This study aimed to investigate the expression of the natriuretic peptide precursor C coding gene nppc and its role in angiogenesis during embryonic period of the zebrafish. Whole mount in situ hybridization was performed to detect the expression pattern of nppc. nppc specific morpholino and nppc mRNA were injected respectively into the one-cell stage embryo to specifically knock-down and rescue the expression of nppc in Tg (flk1:GFP) and Tg (fli1a:nGFP) transgenic lines. The morphology and endothelial cell number of intersegmental vessel (ISV) were analyzed after imaging using the laser scanning confocal microscope. The results revealed that nppc was expressed in the brain, heart and vasculature of zebrafish larvae at 24 and 48 hours post-fertilization (hpf). Knock-down of nppc affected the development of ISV. Endothelial cell number was reduced after the knock-down of nppc. These results suggest that nppc controls zebrafish angiogenesis by affecting the endothelial cell proliferation and migration.
Animals ; Animals, Genetically Modified ; Cell Movement ; Cell Proliferation ; Endothelial Cells ; physiology ; Gene Knockdown Techniques ; Heart ; physiology ; Larva ; Natriuretic Peptides ; genetics ; physiology ; Neovascularization, Physiologic ; RNA, Messenger ; Zebrafish ; genetics ; physiology ; Zebrafish Proteins ; genetics ; physiology

With the application of the photoconversion technology of genetically expressed fluorescent proteins in biologic field, more powerful confocal imaging ability was demanded. The aim of the present study was to establish an experimental model employing confocal simultaneous scanner unit for simultaneous laser stimulation and imaging, taking study of forebrain neurodevelopment in zebrafish as an example. In the present study, 36-48-hour-old Tg(lhx5:kaede) zebrafish embryos were mounted with 1.2% low melting temperature agarose. The forebrain neurons marked with kaede were observed using the simultaneous scanner unit of confocal microscopy. The 405 nm laser was used to stimulate the region of interest (ROI), while 488 and 559 nm lasers were used to acquire images at the same time. The photoconversion state of kaede protein was then reviewed, and the projecting pattern of neurons stimulated by the ultraviolet laser was examined. The results showed that, the fluorescence of stimulated kaede turned from green to red, and the photoconversion of kaede demonstrated anterior dorsal telencephalon (ADt) neurons projected axons ventrally into the anterior commissure (AC) and supraoptic tract (SOT). These results suggest the confocal simultaneous scanner unit meets the demand of the photoconversion experiment. The application of confocal simultaneous scanning technology in examining Tg(lhx5:kaede) zebrafish embryos affords an ideal experimental model in neurodevelopment study.
Animals ; Axons ; physiology ; Microscopy, Confocal ; Neurons ; cytology ; Prosencephalon ; embryology ; Ultraviolet Rays ; Zebrafish ; embryology

We propose that locations of genes on chromosomes can contribute to the prediction of gene regulatory relationships. We constructed a time-based gene regulatory network of zebrafish cardiogenesis on the basis of a spatio-temporal neighborhood method. Through the network, specific regulatory pathways and order of gene expression during zebrafish cardiogenesis were obtained. By comparing the order with locations of these genes on chromosomes, we discovered that there exists a reversal phenomenon between the order and order of gene locations. The discovery provides an inherent rule to instruct exploration of gene regulatory relationships. Specifically, the discovery can help to predict if regulatory relationships between genes exist and contribute to evaluating the correctness of discovered gene regulatory relationships.
Algorithms ; Animals ; Chromosome Mapping ; Chromosomes ; Gene Expression ; Gene Regulatory Networks ; Heart/physiology* ; Zebrafish/genetics*

OBJECTIVETo perform phenotypic identification and characteristic analysis of a new zebrafish mutant 1276 defective in primitive myelopoiesis.

METHODSThe AB strain male zebrafish were mutagenized with N-ethyl N-nitrosourea (ENU) to induce mutations in the spermatogonial cells, and the mutations were transmitted to the offsprings. The F3 embryos were screened by neutral red staining for identifying the mutants defective in primitive myelopoiesis. One of the myeloid mutants 1276 was further studied by cytochemistry and whole mount in stiu hybridization (WISH) with different lineage markers.

RESULTSA total of 2140 mutagenized genomes from the 1296 F2 families were analyzed, and 12 mutants were identified to show abnormal signal by neutral red staining. In the primitive hematopoiesis stage, the mutant 1276 showed the absence of neutral red staining-positive cells in the whole body. The expression of microglia marker apoe was totally lost in the head of the mutant, and the expression of the macrophage marker l-plastin was slightly decreased in the head and remained normal in the ventral dorsal aorta region, but the granulocytes and erythrocytes developed normally. in the definitive hematopoiesis stage, the mutant 1276 still showed abnormal macrophages as found in the primitive hematopoiesis stage, but the granulocytes, erythrocytes and lymphocytes appeared normal.

CONCLUSIONThe zebrafish mutant 1276 shows abnormalities in the function, development and migration of the macrophages in the primitive hematopoiesis stage, which can not be compensated in the definitive hematopoiesis stage.

Animals ; Gene Expression Regulation, Developmental ; Granulocytes ; physiology ; Hematopoiesis ; genetics ; Macrophages ; pathology ; Male ; Mutation ; Myelopoiesis ; genetics ; Zebrafish ; genetics

OBJECTIVETo identify zebrafish mutants with myelopoiesis defects by ENU mutagenesis and large-scale forward genetic screening.

METHODSMale zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in the spermatogonial cells to generate the founders, which were outcrossed with AB to raise F1 fish. The F1 fish from different founders were mated to generate the F2 families. The F3 embryos from F2 sibling crosses were screened by Sudan black B staining and neutral red staining.

RESULTSA total of 350 F2 families from F1 sibling crosses were screened, and 1424 F2 crosses were analyzed. Six mutations were identified resulting in abnormal Sudan black B staining and neutral red staining, indicating the involvement of neutrophil deficiency or macrophage abnormalities.

CONCLUSIONIt is simple and cheap to induce and screen myelopoiesis deficiency in zebrafish by ENU chemical mutagenesis and Sudan black B staining and neutral red staining. These mutants shed light on the identification of the genes important to myelopoiesis in zebrafish.

Animals ; Gene Expression Regulation, Developmental ; genetics ; Genetic Testing ; Male ; Mutagenesis ; Mutation ; Myeloid Progenitor Cells ; physiology ; Myelopoiesis ; genetics ; Zebrafish ; genetics

The grass carp (Ctenopharyngodon idella) collectin gene was cloned from mixed liver and kidney cDNA library. The full length sequence of grass carp collectin was 1128 bp, contained a 5' untranslated region of 229 bp and a 3' untranslated region of 104 bp. The open reading frame of grass carp collectin was 795 bp which could code a 264 amino acids polypeptide, including a terminal codon. Phylogenetic analyses showed that grass carp collectin shared the highest homology with that of zebrafish (Danio rerio). To understand the function of grass carp collectin, we expressed and purified the recombinant protein (P(CRD)) that comprised carbohydrate recognition domain (CRD). Agglutination of Aeromonas hydrophila and Staphylococcus aureus etc. and sugars inhibition experiments showed that: galactose, glucose, mannose and maltose could inhibit the agglutination of Aeromonas hydrophila. Maltose could lower the agglutination of Staphylococcus aureus, whereas peptidoglycan and glucose inhibited it well. In addition, the activity of grass carp collectin could not dependent on Ca(2+).
Amino Acid Sequence ; Animals ; Base Sequence ; Carps ; genetics ; Cloning, Molecular ; Collectins ; genetics ; physiology ; Fish Proteins ; genetics ; physiology ; Molecular Sequence Data ; Phylogeny ; Zebrafish ; genetics