A Robotics-Based Behavioral Paradigm to Measure Anxiety-Related Responses in Zebrafish

Zebrafish are gaining momentum as a laboratory animal species for the study of anxiety-related disorders in translational research, whereby they serve a fundamental complement to laboratory rodents. Several anxiety-related behavioral paradigms, which rest upon the presentation of live predatorial stimuli, may yield inconsistent results due to fatigue, habituation, or idiosyncratic responses exhibited by the stimulus itself. To overcome these limitations, we designed and manufactured a fully controllable robot inspired by a natural aquatic predator (Indian leaf fish, Nandus nandus) of zebrafish. We report that this robot elicits aversive antipredatorial reactions in a preference test and that data obtained therein correlate with data observed in traditional anxiety- and fear-related tests (light/dark preference and shelter-seeking). Finally, ethanol administration (0.25; 0.50; 1.00%) exerts anxiolytic effects, thus supporting the view that robotic stimuli can be used in the analysis of anxiety-related behaviors in zebrafish.     

不同光强对越南抱茎茶光合及荧光特性日变化的影响

为研究不同光强对越南抱茎茶光合作用机理的影响,设置3种处理方式,即全光照(L1)、光强度45%(L2)、光强度15%(L3),分别在遮阴3个月后测定其光合荧光参数的变化。结果表明,不同光强处理下,净光合速率均呈单峰曲线,峰值出现在11:00左右;光合速率下降主要是气孔限制因素引起的;叶绿素荧光参数表明,强光条件下电子传递受阻,热耗散增加,PSII反应中心受到破坏,从而降低了茶花的光合效率;过度遮阴条件下受到光抑制,从而导致光合机构损伤;45%光照条件下光合效率较高。相关性分析表明,净光合速率(Pn)与空气温度(Ta)、相对湿度(RH)、气孔导度(Gs)、蒸腾速率(Tr)、水分利用效率(WUE)、表观电子传递速率(ETR)呈极显著相关,与RH呈极显著负相关,与胞间CO2浓度(Ci)呈显著负相关。影响越南抱茎茶净光合速率的主要环境因子是空气温度和相对湿度。     

Ontogenetic Changes in Daphnia Responsiveness to Fish Kairomone

Exposing Daphnia to the presence of a predator at different stages of ontogenesis leads to different reactions. Predator-induced changes of life history parameters related to the first reproduction schedule, as age and size at first reproduction, take place in the stage preceding that one in which vitellogenesis occurs. Determination of the amount of energy allocated to this first reproduction, including offspring number, occurs in the first 24-h of life. In case of parameters related to reproduction schedule, time since induction until the release of the first clutch of offspring is tens of hours; in case of allocation patterns – more than 100. This suggests that Daphnia do not remember the information of a potential threat but, instead, the decisions on future development are undertaken once they have estimated the reigning predation regime. Shortening of the period during which eggs are carried in the brood chamber, thus shortening the time when Daphnia are more easily visible, may be another adaptation to reduce danger from fish predation and so increasing the probability of survival until the first successful reproduction.     

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.     

绒毛番龙眼对生长光强的形态和生理适应

在100％、50％、25％和8％自然光强下栽培绒毛番龙眼幼苗并研究了其对光环境的适应。100％生长光强下绒毛番龙眼通过增大叶片悬挂角(midrib angle,MA)和比叶重(lamina mass per unit area,LMA),减少叶氮在捕光组分中的分配等降低光能捕获;通过增加类胡萝卜素含量增加热耗散。虽如此,还是发生了比较严重的光抑制,加之叶氮在光合机构中的分配最少,导致光合能力最低,长势最差。8％生长光强下绒毛番龙眼通过降低MA、LMA以及叶片技转,增加叶氮在捕光组分中的分配等提高光能捕获能力,光能转换及利用效率较高,热耗散水平较低,但由于环境光较弱,限制了光合碳同化,植株生长也较慢。50％和25％生长光强下绒毛番龙眼有较强的光能捕获、利用和耗散能力,在几种光处理中长势最好。     

Circadian Rhythms in Stomatal Responsiveness to Red and Blue Light

Stomata of many plants have circadian rhythms in responsiveness to environmental cues as well as circadian rhythms in aperture. Stomatal responses to red light and blue light are mediated by photosynthetic photoreceptors; responses to blue light are additionally controlled by a specific blue-light photoreceptor. This paper describes circadian rhythmic aspects of stomatal responsiveness to red and blue light in Vicia faba. Plants were exposed to a repeated light:dark regime of 1.5:2.5 h for a total of 48 h, and because the plants could not entrain to this short light:dark cycle, circadian rhythms were able to "free run" as if in continuous light. The rhythm in the stomatal conductance established during the 1.5-h light periods was caused both by a rhythm in sensitivity to light and by a rhythm in the stomatal conductance established during the preceding 2.5-h dark periods. Both rhythms peaked during the middle of the subjective day. Although the stomatal response to blue light is greater than the response to red light at all times of day, there was no discernible difference in period, phase, or amplitude of the rhythm in sensitivity to the two light qualities. We observed no circadian rhythmicity in net carbon assimilation with the 1.5:2.5 h light regime for either red or blue light. In continuous white light, small rhythmic changes in photosynthetic assimilation were observed, but at relatively high light levels, and these appeared to be attributable largely to changes in internal CO2 availability governed by stomatal conductance.     

Behavioral and Biochemical Effects of N-Acetylcysteine in Zebrafish Acutely Exposed to Ethanol

Neurochemical Research - Alcohol hangover refers to unpleasant symptoms experienced as a direct consequence of a binge drinking episode. The effects observed in this condition are related to the...     

A Transient Burst of CO(2) from Geranium Leaves during Illumination at Various Light Intensities as a Measure of Photorespiration

A transient CO₂ burst is exhibited by irradiated leaves of the C₃ plant geranium (Pelargonium X hortorum, Bailey) after the irradiance is quickly lowered. The light CO₂ burst appears to be related to photorespiration because of its irradiance dependency and its sensitivity to other environmental components such as CO₂ and O₂ concentration. The term post-lower-irradiance CO₂ burst or PLIB is used to describe the phenomenon. The PLIB appears to be a quantitative measurement of photorespiration with intact geranium leaves. The PLIB has been observed with intact leaves of other C₃ plants but not with C₄ leaves. Therefore, it is proposed that, after maximizing intact leaf photosynthetic rates and leaf chamber gas measuring conditions, photorespiration can be measured with intact C₃ leaves such as geranium as a transient post-lower-irradiance CO₂ burst.     

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10⁶ cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.     

Responsiveness to Retinoic Acid Changes during Chondrocyte Maturation

We previously showed that retinoic acid (RA) participates in the regulation of chondrocyte maturation during endochondral ossification, a process involving multiple developmental stages. To assess whether the responsiveness to RA treatment changes during chondrocyte maturation, immature chondrocytes were isolated from the caudal portion of Day 18-19 chick embryo sterna, a portion that remains cartilaginous through early postnatal life but ossifies with age. The immature cells were allowed to reach different stages of maturation by growth for different time in culture. Progression by the cells toward the mature phenotype during culture was confirmed by increases in average cell diameter, proteoglycan synthesis, and alkaline phosphatase (APase) activity. When developmentally immature passage 0 (PO) cultures were treated with RA (10-100 nM) for 72 h, the cells readily became fibroblastic, reduced drastically their proteoglycan synthesis, and failed to activate type X collagen gene expression. When older cultures (P1 and P2) were treated with RA, the cells acquired a characteristic epithelioid shape and increased their APase activity. Moreover, 5-10% of P1 cells and 20-25% of P2 cells activated type X collagen synthesis in response to RA. RA treatment markedly induced expression of the gene encoding the β isoform of retinoic acid receptor (RARβ) and also provoked a moderate 2.5-fold increase in RARα gene expression. A similar change in responsiveness to RA was observed during maturation in vivo. Chondrocytes were isolated from the cephalic portion of Day 10, 11, 13, and 16 chick embryo sterna, and were treated with different doses of RA (10-100 nM) for 72 h. The cells from the Day 10 sternum failed to activate type X collagen gene expression in response to RA. In contrast, with increasing age of the embryos, an increasing fraction of cells induced type X collagen gene expression in response to RA. We conclude that responsiveness to RA changes during the early stages of chondrocyte maturation and that maturation depends on interactions between exogenous retinoids and the endogenous developmental program of chondrocytes.     

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.¹⁷ that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.     

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.     

Seizures Induced by Pentylenetetrazole in the Adult Zebrafish: A Detailed Behavioral Characterization

Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.     

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.     

Multiplex Cytological Profiling Assay to Measure Diverse Cellular States

Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that “paints the cell” with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar  annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.     

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.