The study was supported by the Basic Research Founding of the Physics Department and the Biology Department of the Naples University Federico II. The authors thank Dr. Claudia Angelini (Institute of Applied Calculus, Consiglio Nazionale delle Ricerche [CNR], Italy) for the statistical support. The authors thank Martina Scanu and Silvia Frassinet for their technical help with collecting the data, and the departmental technicians F. Cassese, G. Passeggio, and R. Rocco for their skillful assistance in the design and realization of the experimental setup. We thank Laura Gentile for helping conducting the experiment during the video shooting. We thank Diana Rose Udel from the University of Miami for shooting the interview statements of Alessandro Cresci.

The following protocol has been approved by the Institutional Animal Care and Use Committee of the University of Naples Federico II, Naples, Italy (2015).
1. Animal Maintenance
<ol>
	<li>Use tanks of at least 200 L to host a shoal of at least 50 individuals of both sexes in each tank. 
	NOTE: The density of the fish in the tank has to be one animal per 2 L or lower. Under these conditions, zebrafish will display normal shoaling behavior.</li>
	<li>Set the maintenance conditions as follow: temperature at 27&#8211;28 &#176;C; conductivity at &#60;500 &#956;S; pH 6.5&#8211;7.5; NO3 at &#60;0.25 mg/L; and a light:dark photoperiod at 10 h:14 h. 
	NOTE: Identical holding conditions must be used for both the mixed population and the separated proactive and reactive populations.</li>
</ol>
2. Personality Selection in Zebrafish
<ol>
	<li>Prepare and place the personality selection tank in a quiet room (Figure 3) with the same water as used in the maintenance tanks.</li>
	<li>Place a video camera above or at the side of the tank. Connect the camera to a computer with a monitor located in an area where there is no visual contact with the tank.</li>
	<li>Select nine fish at random from the maintenance tank and transfer them to the dark side of the personality selection tank, using a knotless net. 
	NOTE: Try to limit the interactions with the tanks and fish to the least amount of time possible. Avoid noise and fast movements. If necessary, transfer the animals in a small volume transporting tank (about 2 L) with water from the holding tank. To avoid air exposure of animals, use a 250 ml beaker and gently induce the animal to enter the beaker. Try to minimize the capture time, avoid collecting multiple fish as it might cause physical damage to the animals and do not hold fish for more than a few seconds in the net as these factors can increase stress. Fish must be fed ad libitum prior to the transfer to the experimental tank. This limits the possibility that different tendencies of food-seeking behavior would affect the behavior of the individuals during the following experiment22. Conduct replicate experiments at the same time of the day. This minimizes variability in the behavior of the experimental groups caused by possible circadian rhythms23.</li>
	<li>After 1 h of acclimatization, open the sliding door. 
	NOTE: Individuals who exit from the hole, exploring the bright side of the tank within 10 min, are considered proactive21.</li>
	<li>After 10 min, gently remove the proactive individuals from the tank and transfer them to the proactive maintenance tank.</li>
	<li>After 15 min, collect the fish that remain in the dark box, which are considered reactive21, and transfer them to the reactive maintenance tank. 
	NOTE: Discard fish that move to the bright side of the tank after 10 min21. Perform the personality test with nine fish at a time until the desired number of proactive and reactive fish necessary for the tests described in section 5 are collected. Consistency of the proactive and reactive personality can be checked regularly using the same approach.</li>
</ol>
3. Set up of the Magnetic Field with the One-dimensional Magnetic Field Manipulation27
<ol>
	<li>Switch on the Power unit (Figure 2A).</li>
	<li>Place the coiled tunnel in the location where the rheotactic protocol will be performed (section 5) but keep it disconnected from the swimming apparatus (Figure 2A). Place a magnetic probe connected with a Gauss/Teslameter inside the tunnel and verify which voltage is necessary to obtain the chosen magnetic field value along the major axis of the tunnel. 
	NOTE: Because of the magnetic properties of a solenoid, the field is reasonably uniform inside the tunnel; this can be checked by slowly moving the probe both horizontally and vertically.</li>
	<li>Disconnect the probe and connect the flow tunnel to the swimming apparatus.</li>
	<li>Start with the rheotactic protocol (section 5).</li>
</ol>
4. Set Up of the Magnetic Field with the Three-dimensional Magnetic Field Manipulation27
<ol>
	<li>Switch on the CPU, DAC, and coil drivers (Figure 2B).</li>
	<li>Set the chosen magnetic field on each one of the three axes (x, y, and z).</li>
	<li>Place the tunnel in the center of the Helmholtz pairs set.</li>
	<li>Start with the rheotactic protocol (section 5).</li>
</ol>
5. Test of the Zebrafish Rheotaxis in the Flow Chamber
<ol>
	<li>Transfer one to five fish to the flow tunnel using a 2 L tank with the sides and the bottom obscured.</li>
	<li>Turn on the pump and set the flow rate in the tunnel to 1.7 cm/s. 
	NOTE: This slow-moving water will keep the water in the tunnel oxygenated and it will facilitate animal recovery.</li>
	<li>Let the animals acclimate to the swimming tunnel for 1 h.</li>
	<li>Start the video recording of the behavior of the fish in the tunnel. 
	NOTE: We used a camera (e.g., Yi 4K Action) with remote control (e.g., Bluetooth) and saved the video as .mpg (30 frames/s).</li>
	<li>Start the stepwise increase of the flow rate according to the chosen experimental protocol (1.3 cm/s in this study; Figure 4). 
	NOTE: For this protocol, we used low flow rates which, for zebrafish, range from 0 to 2.8 BL (body lengths)/s. These flow speeds are in the lower range of flow rates that induce continuous oriented swimming in zebrafish (3%&#8211;15% of critical swimming speed [Ucrit])24. The use of low flow rates (following Brett&#8217;s protocol25) is linked to the speci&#64257;c behavioral characteristics of this species in the presence of water currents. Zebrafish tend to swim along the major axis of the chamber, turning frequently, even in the presence of water &#64258;ow, and tend to swim both upstream and downstream24,26. This behavior is affected by the water &#64258;ow rate, disappearing at relatively high speeds (&#62;8 BL/s)26, when the animals continuously swim facing upstream (full positive rheotactic response). Vertical and transversal displacements are very rare.</li>
	<li>Perform morphometry of the animals (sex and total length [TL], fork length [FL], or BL) on pictures of fish in a morphometric chamber&#8203;.
	<ol>
		<li>Select the appropriate picture.</li>
		<li>Open the picture in ImageJ.</li>
		<li>Take note of the sex of the animal (male zebrafish are slender and tend to be yellowish, while females are more rounded and tend to have blue and white colorings).</li>
		<li>Click Analyze &#62; Set Scale and set the scale of the image in centimeters, using the whole horizontal length of the tunnel as reference.</li>
		<li>Click Analyze &#62; Measure and record the linear length of the animal.</li>
		<li>Calculate its body weight (BW). 
		NOTE: BW is calculated from sex-FL-BW relationships previously built in the lab or from metadata. The whole procedure avoids manipulation stress on the animals.</li>
	</ol>
	</li>
</ol>
6. Video Tracking
<ol>
	<li>Open the video file with Tracker 4.84 Video Analysis and Modeling Tool. 
	NOTE: If necessary, correct any video distortion using perspective and radial distortion &#64257;lters.</li>
	<li>Click on Coordinate system in the upper menu and set the length units to centimeters and the time units to seconds.</li>
	<li>Click on File &#62; Import &#62; Video and open one of the videos in Tracker 4.84.</li>
	<li>Click on &#8220;Coordinate axes&#8221; and set the reference system to track the position of the fish over time, with the x axis along the tunnel. Set the origin at the low corner of the downstream ending wall (at the water outlet).</li>
	<li>Click on Track &#62; New &#62; Point of mass and start tracking one fish at a time. Track the last 5 min of each step that the fish spent at each flow rate.</li>
	<li>Advance the video manually at five-frame intervals (0.5 s) and mark the time and position of the animal at each upstream-downstream turn (UDt; red dots in Figure 5) and at each downstream-upstream turn (DUt; blue dots in Figure 5). 
	NOTE: Use the &#64257;sh eye position as a reference for the &#64257;sh&#8217;s position. Track the animal&#8217;s position using a Point mass. Exclude from the tracking any period of non-oriented swimming (i.e., maneuvering time).</li>
	<li>At the end of each tracking session, select the x-values and time values from the table at the bottom-right corner of the software window. Right-click on the data and click Copy data &#62; Full precision.</li>
	<li>Save the time values and x-values of all turning positions on a template spreadsheet file to calculate the total upstream time (sum of all the intervals between UDts and DUts) and the total downstream time (sum of the intervals between DUts and UDt), as well as the values of the rheotactic index in percentages (RI%) for each flow step (see Figure 5). 
	NOTE: The rheotactic behavior is quantified by the proportion of the total oriented time that the fish spend facing upstream (swimming or rarely freezing [i.e., they stay still at the bottom of the tunnel]27). This proportion is defined as the RI% (Figure 5). 
	<img alt="Equation" src="/files/ftp_upload/59229/59229eq1.jpg" /></li>
</ol>

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G., Struthers, G., Watts, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+Atlantic+salmon+smolts,+Salmo+salar+L.,+through+Loch+Voil,+Scotland.">Tracking Atlantic salmon smolts, Salmo salar L., through Loch Voil, Scotland.</a> Journal of Fish Biology. 19 (5), 519-537 (1981).</li><li>Bottesch, M., 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=A+magnetic+compass+that+might+help+coral+reef+fish+larvae+return+to+their+natal+reef.">A magnetic compass that might help coral reef fish larvae return to their natal reef.</a> Current Biology. 26 (24), R1266-R1267 (2016).</li><li>Boles, L. C., Lohmann, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=True+navigation+and+magnetic+maps+in+spiny+lobsters.">True navigation and magnetic maps in spiny lobsters.</a> Nature. 421 (6918), 60-63 (2003).</li><li>Dingemanse, N. J., Kazem, A. J. N., R&#233;ale, D., Wright, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+reaction+norms:+animal+personality+meets+individual+plasticity.">Behavioural reaction norms: animal personality meets individual plasticity.</a> Trends in Ecology and Evolution. 25 (2), 81-89 (2010).</li></ol>

The authors have nothing to disclose.

The protocol described in this study allows scientists to quantify complex orientation responses of aquatic species resulting from the integration between two external cues (water current and geomagnetic field) and one internal factor of the animal, such as personality. The overall concept is to create an experimental design that allows scientists to separate individuals of different personality and investigate their orientation behavior while controlling separately or simultaneously the external environmental cues.
...

In the present study, we describe a lab-based behavioral protocol which has the scope of investigating the role of fish personality on the orientation response of shoaling fish to external orientation cues, such as water currents and magnetic fields.
The orienting decisions of animals result from weighing various sensory information. The decision process is influenced by the ability of the animal to navigate (e.g., the capacity to select and keep a direction), its internal state (e.g., feeding or reproductive needs), its ability to move (e.g., locomotion biomechanics), and several additional external factors (e.g., time of day, interaction with conspecifics)1.
The role of the internal state or animal personality in the orientation behavior is often poorly understood or not explored2. Additional challenges arise in the study of the orientation of social aquatic species, which often perform coordinated and polarized group movement behavior3.
Water currents play a key role in the orientation process of fish. Fish orient to water currents through an unconditioned response called rheotaxis4, which can be positive (i.e., upstream oriented) or negative (i.e. downstream oriented) and is used for several activities, ranging from foraging to the minimization of energetic expenditure5,6. Moreover, a growing body of literature reports that many fish species use the geomagnetic field for orientation and navigation7,8,9.
The study of rheotaxis and swimming performance in the fish is usually conducted in flow chambers (flume), where fish are exposed to the stepwise increase of the flow speed, from low to high speeds, often until exhaustion (called critical speed)10,11. On the other hand, previous studies investigated the role of the magnetic field in the orientation through the observation of the swimming behavior of the animals in arenas with still water12,13. Here, we describe a laboratory technique that allows researchers to study the behavior of fish while manipulating both the water currents and the magnetic field. This method was utilized for the first time on shoaling zebrafish (Danio rerio) in our previous study, leading to the conclusion that the manipulation of the surrounding magnetic field determines the rheotactic threshold (i.e., the minimal water speed at which shoaling fish orient upstream)14. This method is based on the use of a flume chamber with slow flows combined with a setup designed to control the magnetic field in the flume, within the range of the earth&#8217;s magnetic field intensity.
The swimming tunnel utilized to observe the behavior of zebrafish is outlined in Figure 1. The tunnel (made of a nonre&#64258;ecting acrylic cylinder with a 7 cm diameter and 15 cm in length) is connected to a setup for the control of the flow rate14. With this setup, the range of flow rates in the tunnel varies between 0 and 9 cm/s.
To manipulate the magnetic field in the swimming tunnel, we use two methodological approaches: the first is one-dimensional and the second is three-dimensional. For any application, these methods manipulate the geomagnetic field to obtain specific magnetic conditions in a defined volume of water&#8212;thus, all the values of magnetic field intensity reported in this study include the geomagnetic field.
Concerning the one-dimensional approach15, the magnetic field is manipulated along the water flow direction (defined as the x-axis) using a solenoid wrapped around the swimming tunnel. This is connected to a power unit, and it generates uniform static magnetic fields (Figure 2A). Similarly, in the case of the three-dimensional approach, the geomagnetic field in the volume containing the swimming tunnel is modified using coils of electric wires. However, to control the magnetic field in three dimensions, the coils have the design of three orthogonal Helmholtz pairs (Figure 2B). Each Helmholtz pair is composed of two circular coils oriented along the three orthogonal space directions (x, y, and z) and equipped with a three-axial magnetometer working in closed-loop conditions. The magnetometer works with field intensities comparable with the earth&#8217;s natural field, and it is located close to the geometrical center of the coils set (where the swimming tunnel is located).
We implement the techniques described above to test the hypothesis that the personality traits of the fish composing a shoal influence the way they respond to magnetic fields16. We test the hypothesis that individuals with proactive and reactive personality17,18 respond differently when exposed to water flows and magnetic fields. To test this, we first sort zebrafish using an established methodology to assign and group individuals that are proactive or reactive17,19,20,21. Then, we evaluate the rheotactic behavior of zebrafish swimming in shoals composed of only reactive individuals or composed of only proactive individuals in the magnetic flume tank, which we present as sample data.
The sorting method is based on the different tendency of the proactive and reactive individuals to explore novel environments21. Specifically, we use a tank divided into a bright and a dark side17,19,20,21 (Figure 3). Animals are acclimated to the dark side. When access to the bright side is open, proactive individuals tend to quickly exit the dark half of the tank to explore the new environment, while the reactive fish do not leave the dark tank.

<table><tbody><tr><td>9500 G meter</td><td>FWBell</td><td>N/A</td><td>Gaussmeter, DC-10 kHz; probe resolution:&amp;nbsp; 0.01 &amp;mu;T&amp;nbsp;</td></tr><tr><td>AD5755-1</td><td>Analog Devices</td><td>EVAL-AD5755SDZ</td><td>Quad Channel, 16-bit, Digital to Analog Converter</td></tr><tr><td>ALR3003D</td><td>ELC</td><td>3760244880031</td><td>DC Double Regulated power supply</td></tr><tr><td>BeagleBone Black</td><td>Beagleboard.org</td><td>N/A</td><td>Single Board Computer</td></tr><tr><td>Coil driver</td><td>Home made</td><td>N/A</td><td>Amplifier based on commercial OP (OPA544 by TI)</td></tr><tr><td>Helmholtz pairs</td><td>Home made</td><td>N/A</td><td>Coils made with standard AWG-14 wire</td></tr><tr><td>HMC588L</td><td>Honeywell</td><td>900405 Rev E</td><td>Digital three-axis magnetometer</td></tr><tr><td>MO99-2506</td><td>FWBell</td><td>129966</td><td>Single axis magnetic probe</td></tr><tr><td>Swimming apparatus</td><td>M2M Engineering Custom Scientific Equipment</td><td>N/A</td><td>Swimming apparatus composed by peristaltic pump and SMC Flow switch flowmeter with digital feedback</td></tr><tr><td>TECO 278</td><td>TECO</td><td>N/A</td><td>Thermo-cryostat&amp;nbsp;</td></tr></tbody></table>

assessing the influence of personality on sensitivity to magnetic fields in zebrafish

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as personality. Here, we describe a behavioral protocol designed for the study of the influence of zebrafish personality on their orientation response to multiple external environmental cues, specifically water currents and magnetic fields. This protocol aims to understand whether proactive or reactive zebrafish display different rheotactic thresholds (i.e., the flow speed at which the fish start swimming upstream) when the surrounding magnetic field changes its direction. To identify zebrafish with the same personality, fish are introduced in the dark half of a tank connected with a narrow opening to a bright half. Only proactive fish explore the novel, bright environment. Reactive fish do not exit the dark half of the tank. A swimming tunnel with low flow rates is used to determine the rheotactic threshold. We describe two setups to control the magnetic field in the tunnel, in the range of the earth&#8217;s magnetic field intensity: one that controls the magnetic field along the flow direction (one dimension) and one that allows a three-axial control of the magnetic field. Fish are filmed while experiencing a stepwise increase of the flow speed in the tunnel under different magnetic fields. Data on the orientation behavior are collected through a video-tracking procedure and applied to a logistic model to allow the determination of the rheotactic threshold. We report representative results collected from shoaling zebrafish. Specifically, these demonstrate that only reactive, prudent fish show variations of the rheotactic threshold when the magnetic field varies in its direction, while proactive fish do not respond to magnetic field changes. This methodology can be applied to the study of magnetic sensitivity and rheotactic behavior of many aquatic species, both displaying solitary or shoaling swimming strategies.

As sample data we present results obtained controlling the magnetic field along the water flow direction on proactive and reactive shoaling zebrafish16 using the setup shown in Figure 2A (see section 3 of the protocol). These results show how the described protocol can highlight differences in responses to the magnetic field in fish with different personalities. The overall concept of these trials relies on the finding that the directi...

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Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish

We describe a behavioral protocol designed to assess how zebrafish&#8217;s personalities influence their response to water currents and weak magnetic fields. Fishes with the same personalities are separated based on their explorative behavior. Then, their rheotactic orientation behavior in a swimming tunnel with a low flow rate and under different magnetic conditions is observed.

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as ...

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6.6K Views.  University of Miami. Dit protocol maakt verkenning mogelijk over hoe de interne toestand of persoonlijkheid van een zebravis de oriëntatierespons beïnvloedt op signalen uit de omgeving, zoals waterstromen of magnetische velden. Deze techniek wordt gebruikt om te observeren hoe zowel interne factoren als omgevingssignalen van invloed zijn op de oriëntatie van de zebravissen. Het kan echter ook worden toegepast op een breed scala aan experimentele ontwerpen.Deze methode kan inzicht geven in gedragsecologi...

Video: Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Developmental Biology

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Environment

Behavioral Approaches to Studying Innate Stress in Zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Neuroscience

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Medicine

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Pharmacological Validation of the Prepulse Inhibition of Startle Response in Larval Zebrafish using a Commercial Automated System and Software

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, to the best of our knowledge, all PPI assays performed in the zebrafish have, until now, been done using custom made systems which were only available to individual groups. This has thereby presented challenges, particularly with regard to issues of data reproducibility and standardization. In the present work, we generated a protocol that utilizes commercially available automated systems to pharmacologically validate the PPI&#160;assay in larval zebrafish. Consistent with published findings, we were able to replicate the results of apomorphine, haloperidol and ketamine on the PPI response of 6 days post-fertilization zebrafish larvae.

Here we describe a protocol that utilizes commercially available automated systems to pharmacologically validate the prepulse inhibition (PPI) assay in larval zebrafish.

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish

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