Name: a swimming-induced zebrafish exercise apparatus for versatile training approaches
Uploaded: 2024-10-18T12:50:00
Description: To comprehensively investigate the effects of exercise on health and disease, animal models play a pivotal role. Zebrafish, a widely utilized vertebrate ...

Gratitude is extended to Dr. Omar Mertins for generously providing access to the laboratory for the maintenance of fish and execution of tests. Furthermore, acknowledgments are given to FAPESP (process numbers 2020/12084-8 and 2021/14313-7), CNPq (process number 151756/2022-8), and CAPES for awarding fellowships to support this research.

The procedures received prior approval from the Animal Use Ethics Committee of the Federal University of S&#227;o Paulo (CEUA/UNIFESP no. 9206260521). Only adult female wild-type Danio rerio, aged 6 months and weighing 2.5-3 g, were employed in this study. The equipment and reagents needed for the study are listed in the Table of Materials.
1. Custom-made Zebrafish exercise apparatus
NOTE: The exercise apparatus was custom-bu...

<ol>
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In this study, an innovative, cost-effective exercise system was developed inspired by the swim tunnel respirometer from Loligo Systems21 and the flume system22 for the comprehensive examination of zebrafish swimming performance. The Umax was determined by systematically increasing water flow in discrete stages, with speed increments occurring over short intervals (20-30 min) until the fish reached exhaustion, which was characterized by three consecutive fatigues or the ina...

Physical exercise encompasses any bodily movement performed by skeletal muscles that result in increased energy expenditure, with exercise being a structured and repetitive subset of physical activities1. Exercise, a multifactorial and cost-effective activity involving the entire body, yields numerous health benefits, such as preventing metabolic syndrome and sarcopenia2. Consequently, the field of exercise physiology holds significant interest as it seeks to elucidate how the body adapts to the acute stress of exercise, the chronic stress of physical training, and the overall impact of exercise on health1.
Conducting exercise physiology studies in humans can be both expensive and time-consuming due to challenges in experimental design and participant monitoring3. Therefore, the use of animal models in laboratory settings has been highly recommended due to their genetic and physiological uniformity. Moreover, under controlled laboratory conditions, animals typically have sedentary lifestyles and regulated food intake4. Among animal models, rodents have been the most widely employed in research involving physical exercise1. However, zebrafish (Danio rerio; Hamilton, 1822) is a complementary model to murine and other species for exercise studies5,6,7,8.
In zebrafish research, physical exercise can be conducted using commercially available or custom-built swim tunnels. Among the commercially available options, the Blazka-type tunnel, developed by the Loligo System, is the most frequently utilized7,9,10. This system induces forced swimming through a propeller coupled to an electric motor, generating a continuous water flow within the tunnel. This swimming capability is rooted in the principle of rheotaxis, an innate behavior in fish that drives them to swim against water currents and maintain their position11. Rheotaxis enables the measurement of critical swimming speed (Ucrit), representing the maximum velocity a fish can sustain for a specific duration. However, it is worth noting that this equipment, while valuable for assessing swimming behavior and oxygen consumption, comes with a significant cost12.
Researchers have developed alternative apparatus for exercising zebrafish, often based on the Blazka-type mechanism10,13,14 or simpler mechanisms8,15,16. Nonetheless, these methods may be constrained by the technical demands of the protocol, including extended durations, substantial equipment expenses, and limitations in throughput and precision. Consequently, the primary objective of the study was to design an affordable and user-friendly zebrafish exercise system using readily available materials, providing a new alternative apparatus for physical exercise in fish. A secondary goal was to implement both aerobic and anaerobic exercise regimes in zebrafish, further advancing the utilization of the zebrafish model as an intervention strategy in exercise research.

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			<td>BXV</td>
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			<td>10 k&#937;, 0.25W t</td>
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			<td>Lab Support Stand With Clamp with 30 inch rod&#160;</td>
			<td>Masiye Labs</td>
			<td>RSC0001</td>
			<td>Support the horizontal pipes</td>
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			<td>LCD screen&#160;</td>
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			<td>Perforated Circuit Board single sided</td>
			<td>KY WIN ROBOT</td>
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			<td>5 x 10 cm</td>
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			<td>Potentiometer</td>
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			<td>DL-ALPSA01</td>
			<td>10k&#937;</td>
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			<td>One World</td>
			<td>5603131000</td>
			<td>To avoid leaks</td>
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			<td>Silicone hose</td>
			<td>Tigre</td>
			<td>14211250</td>
			<td>2 cm inner</td>
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			<td>QHTITEC</td>
			<td>EU/US PLUG</td>
			<td>Arduine system welding&#160;</td>
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			<td>Solder Wire Spool</td>
			<td>BEEYIHF</td>
			<td>I001-A001-Set</td>
			<td>Arduine system welding&#160;</td>
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			<td>Threaded Male Socket and Unthreaded Female Socket CPVC Pipe Fitting</td>
			<td>TIgre</td>
			<td>35447849</td>
			<td>3/4-inch&#160;</td>
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			<td>Tricaine (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Anesthetic</td>
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			<td>UNO-R3 board UNO R3 CH340G+MEGA328P Chip 16Mhz&#160;</td>
			<td>FSXSEMI</td>
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			<td>For Arduino UNO R3 Development board</td>
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			<td>Unthreaded CPVC Tee Pipe Fitting, Female</td>
			<td>Tigre</td>
			<td>22200267</td>
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			<td>Unthreaded Female CPVC Socket Pipe Fitting</td>
			<td>Tigre</td>
			<td>22170260</td>
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			<td>Water Flow Sensor&#160; model YF-B5&#160;</td>
			<td>Siqma Robotics</td>
			<td>SQ8659</td>
			<td>1-25 L/min</td>
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			<td>Water Pump&#160;</td>
			<td>Sunsun</td>
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			<td>Model HJ-2041, 3000L/h, 65W</td>
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			<td>Water reservoir</td>
			<td>Custom</td>
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a swimming-induced zebrafish exercise apparatus for versatile training approaches

To comprehensively investigate the effects of exercise on health and disease, animal models play a pivotal role. Zebrafish, a widely utilized vertebrate model organism, offers a unique platform for such studies. This study introduced the development of a cost-effective apparatus tailored for zebrafish exercise studies utilizing readily available materials. The device is founded on the principles of a swim tunnel and encompasses a network of pipes and valves linked to a submersible pump. Water flow is meticulously monitored by a sensor and regulated via valves. To assess the apparatus&#39;s effectiveness, two training protocols were implemented: moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT). Fish were trained collectively, and their swimming performance was assessed through an endurance test. Both training protocols led to improvements in swimming performance following 30 days of training and induced alterations in the molecular response to exercise compared to a sedentary control group. Notably, HIIT demonstrated superior efficiency over MICT. The zebrafish training system proved to be a valuable tool for investigations in exercise physiology and further advances the utility of the zebrafish model in this field.

The exercise apparatus demonstrated remarkable efficiency in regulating flow velocity. To gradually enhance swimming speed, water flow was incrementally increased weekly for all groups, except for the SED group, which was maintained at a constant flow velocity of 0.06 m/s. Notably, the apparatus allowed for a remarkable level of precision, achieving flow velocity adjustments as fine as 0.001 m/s. However, the error rate was 30% at low velocities, such as 0.06 m/s. At high velocities, such as 0.3 m/s and 0.5 m/s, the erro...

The exercise apparatus designed for less fortunate fish facilitates the implementation of diverse exercise protocols with varying intensities by manipulating water flow speed, achievable through rheotaxis.

A Swimming-Induced Zebrafish Exercise Apparatus for Versatile Training Approaches

To comprehensively investigate the effects of exercise on health and disease, animal models play a pivotal role. Zebrafish, a widely utilized vertebrate ...

In zebrafish research, physical exercise can be conducted using commercially-available or custom-built swim tunnels. Here, we developed a high-throughput, user-friendly exercise system for zebrafish, using materials that are easily available. The setup is based on the real access behavior of zebrafish.To test the efficiency of the apparatus, two exercise protocols were employed. Moderate-intensity continuous training, and high-intensity interval training. To monitor the water flow speed indicated by the flow sensor, we employ an Arduino Nano, a 16-by-2 LCD screen, a 10-kiloohm one-quarter watt through-hole resistor, and a 10-kiloohm potentiometer.Assemble the components following the provided scheme. Using a perf board, solder wire, solder iron, wire, and three male jumpers. Load the provided Arduino sketch using an appropriate USB cable via the Arduino IDE.Water flow rate, time since the last reset in minutes, and velocity of water are displayed on the LCD screen. To assemble the water recirculation system, use three-quarter inch PVC pipes and three-quarter inch connectors. It is necessary to create two water outlets.The first will lead water from the main flow back to the reservoir, and the second will direct the flow to the fish training area. Add a global valve to regulate the return flow. Add an upper water outlet valve to regulate the water flow to the training area.Attach a T-connection to the upper valve. Note that it is necessary to add a net inside the connection near the valve to prevent fish from entering the bottom part of the apparatus. In the T-connection, add a global valve, which will serve as a gate for the entry and exit of fish from the system.Attach the acrylic pipe to the T-connector. Next, attach the flow sensor to the acrylic pipe. Note that it is necessary to place a screen inside the pipe to prevent fish from passing through.Connect the partially-assembled apparatus to the submersible pump inside the water reservoir. To maintain water recirculation, it is necessary to connect the hose to the final portion of the apparatus after the flow sensor. Finally, connect the flow sensor to the monitor for monitoring water flow velocity.Note that these are connectors that must be connected in the correct order. Now it is ready to use. Before adding the zebrafish to the apparatus, it is necessary to set the flow.To do this, first open both flow control valves completely to allow bubbles to exit the system, keeping the fish inlet and outlet valve closed. Next, adjust the flow to maximum speed, approximately 0.6 meters per second. This will provide greater precision in flow adjustment using the upper valve.Capture the fish, and then close the upper valve to stop the water flow. Then fully open the next valve that will serve as the fishes'entry gate. It is important to keep the valve opening facing upwards.Immediately afterwards, close the inlet valve and open the flow control valve. It is possible to place a small group of up to six fishes, which should be inserted all at once. To remove the fishes, completely close the upper valve, rotate the fish inlet outlet valve, turning its opening downward.Then fully open the valve. Use a container to collect the fish, along with the water that will flow by gravity from the apparatus. Finally, close the inlet outlet valve to stop the water leakage.Before exercise protocols are employed, it is necessary to acclimate the animals to a relatively low and constant speed for at least 60 minutes per day for one week. Before each exercise session, it is necessary to acclimate the animals for 10 minutes. After the acclimatization period, Umax needs to be determined by assessing the maximum swimming speed against the flow.For this, the flow rate will be increased by 0.02 meters per second, every minute, until the fish reaches exhaustion. To increase the speed, simply open the upper valve gently, monitoring the speed through the LCD. Subject the fish to forced swimming against the water flow at 60%of Umax, as determined in the maximal capacity test, for 35 minutes.Note during the first 10 minutes, the fish is acclimated to the same speed as the sedentary group, 0.06 meters per second. The fish swim against the constant water flow rate without reaching a state of physical exhaustion. Subject the fish to forced swimming, alternating between swimming speeds.Two minutes at 90%of Umax, followed by two minutes at 30%of Umax, repeated for 18 minutes, nine cycles. The fish swim against an alternate water flow rate without reaching a state of physical exhaustion. The setup was precise, allowing fine adjustments in flow velocity.However, the error was around 30%when the speed was low, 0.06 meters per second. When the speed was high, around 0.3 meters per second and 0.5 meters per second, the error rate was only between three and four percent. The fastest speeds reached during training was 0.4 meters per second in the sedentary group, 0.44 meters per second in the moderate training group, and 0.49 meters per second in the high-intensity group in the final endurance test.Both training regimens were designed to cover the same distance, but the high-intensity training protocol resulted in quicker performance improvements, as evidenced by the weekly Umax increase. The fishes exposed to high-intensity training improved their performance by 10%each week, totaling around 30%overall. On the other hand, the fishes exposed to moderate training displayed a slower progress, with a noticeable 10%increase in Umax only in the third week, and no further improvement in the following week.These results highlight how the choice of training protocols may affect zebrafish physical performance differently. In this study, we created a new cost-effective exercise system inspired by existing swim tunnel and flume systems. This system allows for a thorough examination of zebrafish swimming performance.We determined Umax by gradually increasing water flow until the fish were exhausted, characterized by three consecutive fatigues or the inability to keep swimming against the current. These findings helped us to design two exercise protocols and confirm the effectiveness of our swim tunnel apparatus for assessing zebrafish physical performance. Additionally, this compact system is versatile, covering a wide range of water flow speeds, and making it easy to customize training protocols.

a-swimming-induced-zebrafish-exercise-apparatus-for-versatile

Research

JoVE Journal

Biology

Experimental Approaches to Tissue Engineering

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

We have developed a simple and versatile system to feed hard ticks on laboratory rabbits. Our non-laborious protocol uses easily accessible materials and can be adjusted depending on the requirements of the various experimental settings. The method allows comfortable monitoring and/or sampling of ticks during the entire feeding period.

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...