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-built. For details, see Figure 1, Supplementary Table 1, Supplementary File 1, and Supplementary File 2.
<ol>
	<li>Place a submersible pump (N) inside a water tank (O) (&#8805;30 L). Ensure that the water meets the following conditions: pH of 7.2 &#177; 0.5 and 400 &#177; 50 &#181;S, 28 &#177; 1 &#176;C.</li>
	<li>Flowing Supplementary Table 1 and Figure 1, connect Pipe (I) to point Tee Pipe (B), and attach a small pipe (G) to the side of B. From G, establish connections to Globe Valve (F), then to another G, and in sequence to Pipe Elbow (A) and I, thereby completing the segment responsible for regulating water pressure within the system. This regulation is achieved through a return flow to the water tank (O).</li>
	<li>In the alternate section of B, link it to a Pipe (J), followed by connections to A and G. Utilize Socket Pipe Fitting (D) to connect the Gate Water Valve (E) to G.</li>
	<li>Integrate a fish entry port into the system by connecting B to G at one end and attaching another G at the opposite end. Subsequently, connect Socket Pipe Fitting (C) to this second G, establishing a sequence that connects to the Acrylic Pipe (K), which is crucial for visualizing swimming behavior.
	<ol>
		<li>To link K to the Water Flow Sensor (M), utilize pipes C, G, and D. Proceed to connect M to G using D, and then integrate A, G, and H to facilitate the water&#39;s return to the reservoir. 
		NOTE: Insert a mosquito screen in the short pipe segment between the gate and globe valve (F2) to prevent fish from accessing other sections of the apparatus. The globe valve (F) serves a dual purpose. The first globe valve (F1) controls the water flow returning to the reservoir before entering the remainder of the apparatus, acting as a system pressure control valve. The globe valve (F2) is an entry and exit point for the zebrafish within the system.</li>
	</ol>
	</li>
	<li>Attach a water flow sensor downstream of the acrylic pipe. 
	NOTE: The flow sensor should be connected to an LCD display and programmed using an Arduino (Figure 1). Details of the Arduino setup are provided in Supplementary File 2.</li>
</ol>
2. Apparatus operation
<ol>
	<li>To safely introduce the fish into the system, it is essential to interrupt the water flow. To achieve this, close the Gate Valve (E) while keeping the Globe Valve (F1) open. Subsequently, open the Globe Valve (F2), which serves as the entrance to the system, gently introduce the fish, and promptly close the F2 valve. Finally, open the E valve to fill the exercise area with water.</li>
	<li>Use the globe valve to control flow speed, diverting water back to the reservoir when needed.</li>
	<li>Utilize the gate valve (F2) for precise flow adjustment and to manage fish access.</li>
	<li>To remove the fish at the end of the test, close valve (E) upon observing exhaustion criteria. Then, open valve F and rotate it 180&#176; relative to the axis of the acrylic tube; this will facilitate the drainage of water carrying the exhausted fish along with it.</li>
	<li>Perform the flow monitoring. 
	NOTE: It is necessary to monitor the water flow speed through a system that incorporates an Arduino Nano, a 16 x 2 LCD screen, a 10 k&#937;, 0.25 W through-hole resistor, and a 10 k&#937; potentiometer. The flow sensor continuously monitors water flow speed based on Hall Effect technology17. Each pulse of current corresponds to one revolution of the sensor flopper, resulting in a frequency (Hz) of 6.6 x Q (flow rate in L/min).
	<ol>
		<li>Connect the appropriate wires of the flow sensor to the 5 V, GND, and D2 pins of the Arduino Nano (Supplementary Table 1). Load the provided sketch (Supplemental File 1) into the Arduino using the Arduino IDE. Power the system through the Arduino USB port. 
		NOTE: The flow measurements are displayed on the 16 x 2 LCD screen. The calibration of the water flow sensor is depicted in Figure 2. The schematics of the Arduino connections to the LCD are illustrated in Figure 3.</li>
	</ol>
	</li>
</ol>
3. Endurance test
NOTE: This step outlines the procedure for the Endurance test to determine the maximum swimming speed (Umax) of zebrafish.
<ol>
	<li>First, allow the fish to adapt for 60 min per day to a low water flow speed (0.06 m/s) inside the swimming tunnel for two weeks. 
	NOTE: After a 24 h preconditioning period, individual zebrafish will undergo the sustained swimming performance test. The purpose of this test is to establish the Umax of each fish.</li>
	<li>Place the zebrafish individually in the apparatus.</li>
	<li>Testing conditions: Position the fish against a water flow with an initial speed of 0.06 m/s for 10 min.</li>
	<li>Velocity Increments: Increase the water flow in discrete stages, with velocity increments of 0.02 m/s occurring every minute for 40-50 min.</li>
	<li>Umax determination: Recpod the maximum swimming speed (Umax) when the fish meet the exhaustion criteria. 
	NOTE: Exhaustion is defined when the first of the following situations is observed: (1) Inability to maintain its position against the water flow for more than three instances, or (2) Inability to sustain its position for longer than 5 s.</li>
	<li>Close the valve (E) upon observing exhaustion criteria. Then, open valve F and rotate it 180&#176; relative to the axis of the acrylic tube. This will facilitate the drainage of water, carrying the exhausted fish.</li>
</ol>
4. Exercise groups and procedure
NOTE: To establish distinct exercise protocols, it is essential to include a sedentary group exposed to identical experimental conditions to compare the effects of exercise protocols, albeit without undergoing high-intensity exercise. It is also essential to establish the Umax because the fractions of the Umax value are necessary to determine the intensity of exercise protocols.
<ol>
	<li>Sedentary (SED) group: Subject the fish to forced swimming against the water flow at 0.06 m/s for 60 min. 
	NOTE: The apparatus generates a continuous water flow, compelling the fish to swim against this current based on the principle of rheotaxis11.</li>
	<li>Moderate-Intensity Continuous Training (MICT) group: Subject the fish to forced swimming against the water flow at 60% of Umax, as determined in the maximal capacity test, for 35 min. 
	NOTE: This protocol was adapted from H&#250;ngaro et al.18. During the first 10 min, the fish was acclimated to the same speed as the sedentary group (0.05 m/s).</li>
	<li>High-Intensity Interval Training (HIIT) group: Subject the fish to forced swimming alternating the swimming speeds: 2 min at 90% of Umax followed by 2 min at 30% of Umax, repeated for 18 min (9 cycles). This protocol was adapted from Marcinko et al.19. 
	NOTE: During the first 10 min of the exercise period, it is necessary to acclimate the fish to the same speed as the sedentary group (0.06 m/s).</li>
	<li>Implement all exercise protocols for 5 days a week over a four-week period. 
	NOTE: Fish should be housed in aquariums that provide suitable conditions, and they should only be introduced into the exercise apparatus during designated exercise periods. Fish should be provided with tropical fish-flaked food three times daily, and the water in maintenance aquariums should undergo a partial change every 2 days.</li>
	<li>Repeat the endurance test at the end of each week, with latency and velocity data at the point of fatigue as indicators of physical conditioning parameters.</li>
	<li>To induce overtraining effects, increase the water flow velocity weekly based on the results of the endurance test conducted after each 4-day cycle of training. Training durations must be adjusted to account for the distance traveled (velocity &#215; time), and these durations should remain consistent between the exercised groups.</li>
	<li>Adjust the swimming time in response to the increased water flow, thereby standardizing the training load across the exercised groups.</li>
</ol>
5. Body measurements
<ol>
	<li>Anesthetize the fish with 0.0075% tricaine (w/v) by immersion for conducting body measurements (weight and size)20.</li>
	<li>Photograph and weigh the fish to determine body dimensions using ImageJ software.</li>
	<li>Express the data in terms of Body Condition Indices (weight [g]/standard length [mm]2; BMI) and Body Condition Scoring (BCS)20.</li>
	<li>To eliminate size and weight variations caused by egg formation, subject the fish to standard breeding20, followed by measurements and weighing.</li>
</ol>

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming-induced+exercise+promotes+hypertrophy+and+vascularization+of+fast+skeletal+muscle+fibres+and+activation+of+myogenic+and+angiogenic+transcriptional+programs+in+adult+zebrafish.">Swimming-induced exercise promotes hypertrophy and vascularization of fast skeletal muscle fibres and activation of myogenic and angiogenic transcriptional programs in adult zebrafish.</a> BMC Genomics. 15 (1), 1-20 (2014).</li><li>Blazina, A. R., Vianna, M. R., Lara, D. 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It is essential to clarify that there are no competing financial interests associated with the research presented in this manuscript. No financial partnerships or affiliations with organizations or entities have been established that could potentially influence or bias the outcomes of this work. This statement serves as an assurance that the research process was straightforward and honest, with no financial conflicts influencing the results. The presentation of this work is motivated by a genuine passion for the subject matter, driven solely by a love for academia and the pursuit of scientific knowledge.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CPVC Female 90-Degree Elbow for Plumbing</td>
			<td>Tigre</td>
			<td>22150260</td>
			<td>3/4-inch&#160;</td>
		</tr>
		<tr>
			<td>24AWG Wire</td>
			<td>Sky Cablo Store</td>
			<td></td>
			<td>Connection between components in the Perforated Circuit Board (1m)</td>
		</tr>
		<tr>
			<td>Acrylic pipe</td>
			<td>The Clear Plastic Shop</td>
			<td>41138408</td>
			<td>3/4-inch&#160;</td>
		</tr>
		<tr>
			<td>Aquarium Submersible Fish Tank</td>
			<td>Aqua Tank</td>
			<td></td>
			<td>300w</td>
		</tr>
		<tr>
			<td>CPVC Pipe</td>
			<td>Tigre</td>
			<td>10121787</td>
			<td>3/4-inch&#160;</td>
		</tr>
		<tr>
			<td>Female Threaded Gate Water Valve</td>
			<td>Tigre</td>
			<td>27950310</td>
			<td>3/4-inch&#160;</td>
		</tr>
		<tr>
			<td>Female Threaded Globe Water Valve</td>
			<td>Tigre</td>
			<td>27940510</td>
			<td>3/4-inch&#160;</td>
		</tr>
		<tr>
			<td>hrough-hole resistor</td>
			<td>BXV</td>
			<td></td>
			<td>10 k&#937;, 0.25W t</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>LCD screen&#160;</td>
			<td>Eichip</td>
			<td></td>
			<td>16 x 2, model JHD162A</td>
		</tr>
		<tr>
			<td>Male x Male Dupont Jumpers</td>
			<td>Chyan</td>
			<td></td>
			<td>Connection between arduino and flow sensor (30 cm)</td>
		</tr>
		<tr>
			<td>Perforated Circuit Board single sided</td>
			<td>KY WIN ROBOT</td>
			<td></td>
			<td>5 x 10 cm</td>
		</tr>
		<tr>
			<td>Potentiometer</td>
			<td>LUSYA</td>
			<td>DL-ALPSA01</td>
			<td>10k&#937;</td>
		</tr>
		<tr>
			<td>Roll of Water Blocking Tape</td>
			<td>One World</td>
			<td>5603131000</td>
			<td>To avoid leaks</td>
		</tr>
		<tr>
			<td>Silicone hose</td>
			<td>Tigre</td>
			<td>14211250</td>
			<td>2 cm inner</td>
		</tr>
		<tr>
			<td>Solder Station</td>
			<td>QHTITEC</td>
			<td>EU/US PLUG</td>
			<td>Arduine system welding&#160;</td>
		</tr>
		<tr>
			<td>Solder Wire Spool</td>
			<td>BEEYIHF</td>
			<td>I001-A001-Set</td>
			<td>Arduine system welding&#160;</td>
		</tr>
		<tr>
			<td>Threaded Male Socket and Unthreaded Female Socket CPVC Pipe Fitting</td>
			<td>TIgre</td>
			<td>35447849</td>
			<td>3/4-inch&#160;</td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>UNO-R3 board UNO R3 CH340G+MEGA328P Chip 16Mhz&#160;</td>
			<td>FSXSEMI</td>
			<td></td>
			<td>For Arduino UNO R3 Development board</td>
		</tr>
		<tr>
			<td>Unthreaded CPVC Tee Pipe Fitting, Female</td>
			<td>Tigre</td>
			<td>22200267</td>
			<td>3/4-inch&#160;</td>
		</tr>
		<tr>
			<td>Unthreaded Female CPVC Socket Pipe Fitting</td>
			<td>Tigre</td>
			<td>22170260</td>
			<td>3/4-inch&#160;</td>
		</tr>
		<tr>
			<td>Water Flow Sensor&#160; model YF-B5&#160;</td>
			<td>Siqma Robotics</td>
			<td>SQ8659</td>
			<td>1-25 L/min</td>
		</tr>
		<tr>
			<td>Water Pump&#160;</td>
			<td>Sunsun</td>
			<td></td>
			<td>Model HJ-2041, 3000L/h, 65W</td>
		</tr>
		<tr>
			<td>Water reservoir</td>
			<td>Custom</td>
			<td></td>
			<td>30 L</td>
		</tr>
	</tbody>
</table>

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 ...

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

Research

JoVE Journal

Biology

1.0K Views.  Federal University of São Paulo. 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.

Video: A Swimming-Induced Zebrafish Exercise Apparatus for Versatile Training Approaches

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 ...