We thank the Washington University Zebrafish Shared Resource for animal care. This research was supported by the NIH (R01 NS113915 to M.H.M.).

Adult zebrafish of the Ekkwill and AB strains were maintained at the Washington University Zebrafish Core Facility. All animal experiments were performed in compliance with IACUC institutional animal protocols.
NOTE: An example of the experimental setup is shown in Figure 1A. The calibration lid (customized), swim endurance lid (customized), and swim behavior lid (standard, enclosed tunnel lid) are shown in Figure 1B. The experimental...

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Adult zebrafish are a popular vertebrate system for modeling human diseases and studying mechanisms of tissue regeneration. CRISPR/Cas9 genome editing has revolutionized reverse genetic studies for modeling disease in zebrafish; however, large-scale genetics in adult zebrafish has been hindered by biological and technical challenges, including the unavailability of adult zebrafish tissues to high-throughput phenotyping. Given the complex anatomy of adult zebrafish, prolonged histological processing is required to obtain ...

Adult zebrafish are eminently used to investigate mechanisms of neuromuscular and musculoskeletal development and disease modeling1,2,3. Zebrafish are capable of efficient, spontaneous repair of multiple tissues, including the brain, spinal cord, and skeletal muscle4,5,6,7. The remarkable capacity to regenerate neuromuscular tissues and model diseases is attracting a growing scientific community into adult zebrafish research1,2,3. However, while assays of locomotion and swim behavior are available and standardized for larval zebrafish, there is a growing need to develop analogous protocols in adult fish8,9,10,11. The goal of this study is to describe protocols to quantify swim endurance and swim behavior in adult zebrafish. We present these protocols in the context of spinal cord regeneration research. However, the behavioral protocols described here are equally applicable to studies of neural and muscle regeneration, neuromuscular and musculoskeletal development, as well as neuromuscular and musculoskeletal disease modeling.
Zebrafish reverse paralysis within 8 weeks of complete spinal cord transection. Unlike poorly regenerative mammals, zebrafish display pro-regenerative immune, neuronal, and glial injury responses that are required for functional spinal cord repair12,13,14. An ultimate readout of functional spinal cord repair is the ability of the lesioned tissue to regain its function after injury. A suite of standardized methods to assess functional regeneration in rodents include locomotor, motor, sensory, and sensorimotor tests15,16,17. Widely used tests in mouse spinal cord injury include the locomotor Basso Mouse Scale (BMS), forelimb motor tests, tactile sensory tests, and grid walking sensorimotor tests15,17. In contrast with mammalian or larval zebrafish systems, behavioral tests in adult zebrafish are less developed, yet much needed to accommodate the growing needs of the tissue regeneration and disease modeling communities.
Complete spinal cord transections result in complete paralysis caudal to the injury site. Shortly after the injury, paralyzed animals are less active and avoid swimming as much as possible. To compensate for lost swim capacity, paralyzed animals display short, frequent bursts by overusing their pectoral fins, which lie rostral to the lesion. This compensatory swim strategy results in rapid exhaustion and lower swim capacity. As the zebrafish spinal cord regenerates, animals regain a smooth oscillatory swim function caudal to the lesion, allowing for increased swim endurance and improved swim behavior parameters. Here, we describe methods to quantify zebrafish swim endurance at increasing water current velocities and swim behavior at low current velocities.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AutoSwim software</td>
			<td>Loligo Systems</td>
			<td>MI10000</td>
			<td>Optional - for Automatic control of current velocity</td>
		</tr>
		<tr>
			<td>Customized lid</td>
			<td>Loligo Systems</td>
			<td>MI10001</td>
			<td>This customized lid is used for swim endurance</td>
		</tr>
		<tr>
			<td>DAQ-BT</td>
			<td>Loligo Systems</td>
			<td>SW10600</td>
			<td>Optional - for Automatic control of current velocity</td>
		</tr>
		<tr>
			<td>Eheim pump</td>
			<td>Loligo Systems</td>
			<td>PU10160</td>
			<td>20 L/min. This pump is placed in theflow-through tank.</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>Fiji</td>
			<td>Freely available through Image J (Fiji)</td>
			<td>Specific script available at https://github.com/MokalledLab/SwimBehavior</td>
		</tr>
		<tr>
			<td>Flowtherm</td>
			<td>Loligo Systems</td>
			<td>AC10000</td>
			<td>Handheld digital flow meter - for calibration</td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>Loligo Systems</td>
			<td>VE10380</td>
			<td>USB 3.0 color video camera (4MP)</td>
		</tr>
		<tr>
			<td>IR light panel</td>
			<td>Loligo Systems</td>
			<td>VE10775</td>
			<td>450 x 210 mm, placed under the swim tunnel&#160; chamber</td>
		</tr>
		<tr>
			<td>Monofocal lens</td>
			<td>Loligo Systems</td>
			<td>VE10388</td>
			<td>25mm manual lens</td>
		</tr>
		<tr>
			<td>PVC Tubing</td>
			<td>VWR</td>
			<td>60985-534</td>
			<td>5/16 x 7/16&#34;&#160; Wall thickness: 1/16&#34;</td>
		</tr>
		<tr>
			<td>R Studio</td>
			<td>R Studio</td>
			<td>Freely available. Version 3.6 with extra packages.</td>
			<td>Specific script available at https://github.com/MokalledLab/SwimBehavior</td>
		</tr>
		<tr>
			<td>Swim tunnel respirometer</td>
			<td>Loligo Systems</td>
			<td>SW10060</td>
			<td>5L (120V/60Hz). The system includes the swim chamber, motor, manual control of water current velocity, 1 pump placed inside the chamber, standard swim tunnel lid for swim behavior, and modified swim tunnel lid for calibration</td>
		</tr>
		<tr>
			<td>uEye Cockpit</td>
			<td>IDS</td>
			<td>Freely available software to control camera parameters</td>
			<td>Alternative cameras and accompanying softwares could be used</td>
		</tr>
		<tr>
			<td>Vane wheel flow probe</td>
			<td>Loligo Systems</td>
			<td>AC10002</td>
			<td>Digital flow probe - for calibration</td>
		</tr>
	</tbody>
</table>

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.

We set up the swim tunnel as described in section 1 of this protocol (Figure 1). We assessed the swim endurance (section 2 of this protocol) as well as swim behavior (sections 3 and 4 of this protocol) of adult zebrafish at baseline and after spinal cord injury (Figure 2).
For establishing baseline motor function, we examined the swim endurance of wild-type zebrafish under increasing water current velocities (Figu...

Watch this Scientific Journal Video about Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish at JoVE.com

Assessment of Swim Endurance and Swim Behavior 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. ...

Zebrafish offer a valuable neural regeneration model because of their ability to recover from spinal cord injury. This method describes swim endurance and swim behavior as readouts of functional spinal cord repair. This technique provides reliable and quantifiable assessment of swim endurance and swim behavior in adult zebrafish.These methods are equally applicable to studies of neuromuscular and musculoskeletal development, disease, and regeneration. To assess swim endurance, open the flow velocity control software. Click on the box labeled Experiment and uncheck Uswim and Uwater.Then change the flow speed in the Uwater box on the bottom left to adjust water current velocities. To begin an automated protocol, click on the Start logging box. In the dialogue window that opens, choose Automated from the dropdown list.To open a previously saved protocol file, click on the file icon next to the protocol file. Next, set up the output file by clicking on the file icon next to the log file. In the file explorer window that opens, name the output file and save it in the desired location.Then, set up a split lap timer window to ensure simultaneous access to the flow velocity control software and timer windows on the computer screen. Next, set up a fish collection tank to house the exhausted fish after their removal from the swim tunnel. Fill the collection tank and a long polyvinyl chloride tube with zebrafish system water.Place one end of the prefilled tube in the collection tank and another in the buffer tank, ensuring that water can freely flow from the buffer tank into the collection tank. Clamp the upper end of the tube with a binder clip to prevent water flow, and use the binder clip to control the outflow of water as needed. Close the swim tunnel with the swim endurance lid before placing one group of fish inside the swim tunnel.To acclimate the fish to the swim tunnel and flow direction, start the split lap timer and adjust the current velocity to zero centimeters per second for the first five minutes, nine centimeters per second for the next five minutes, and 10 centimeters per second for the following five minutes. Following the acclimation, open the protocol and start the automated flow velocity control program, which will increase the water current velocity by two centimeters per second every minute. Monitor the fish for exhaustion.Exhausted fish will be pushed toward the back end of the swim tunnel. To ensure a fish is exhausted, gently tap the back end of the tunnel or create a shadow over that area to stimulate the fish to swim. Exhausted fish did not respond to the startle stimulus and lay flat at the back end of the tunnel.When a fish is exhausted, unclamp the fish collection tube. Open the swim tunnel window and collect the fish in the collection tank. Record the time at exhaustion using the split lap timer.After all the fish are exhausted and collected in the collection tank, click on the Emergency Stop button on the flow velocity control software and stop the timer. To capture movies for the swim behavior assay, place a group of fish in the swim tunnel and close the tunnel using a standard, fully-enclosed lid. Then, open a new recording window and name the file.Do not click Record yet. Before beginning a new experiment, place a paper towel or piece of fabric on the side of the swim tunnel to ensure all behaviors are due to fish swimming, and not due to a startle response caused by movement in the environment. After ensuring that the water is calm and no ripples are moving across the frame, click on Record in the camera software window to start recording the movie file.Then click on Start in the flow velocity control software to begin the protocol, which will continue uninterrupted. Watch the movie to ensure that no frames are dropped, there are no bubbles in the field-of-view, and all fish are recorded. Once the movie recording is completed, click on Emergency Stop to end the flow velocity control protocol and check that the data output file is saved automatically.Then, close the recording window to save the movie file. After the recording, remove the lid. Carefully retrieve the fish and return them to their tank.To analyze the captured movies, open the tracking_v2. ijm script in Fiji and click on Run to begin the program. In the popup window, choose the folder containing the swim behavior movies to track and click on Open.Look for frame one of the first movie, a dialogue box, and the region-of-interest, or ROI manager, that will come up. Follow the directions given in the dialogue box and create an ROI at the bottom of the swim tunnel chamber. Then click on OK and ensure that no black corners are seen.The threshold window will open along with an edited thresholded frame one. Change the color scheme from black and white to red and adjust the max value until frame one only shows the fish in red and nothing else. Record the threshold and click OK in the dialogue box.For aligning, assembling, and acquire descriptive statistics, open the SwimBehavior_v7-3. R script in RStudio and click on Source in the top right corner of the script section. In a new window that opens, choose the folder containing the _raw.csv files generated by Fiji and click Open. The program will run automatically. In the popup dialogue box, confirm the number of fish in each movie.Click on Yes if the given numbers are correct or no if the numbers are incorrect. Once the files are aligned, check for a newly-generated _aligned. csv file.After ensuring that the program combines the data, runs statistics, and plots output graphs, check for the analysis files generated in a new folder labeled Results within the parent folder containing the _raw. csv and _aligned. csv files.Swim endurance assessed at two, four, and six weeks post-injury showed a 60%loss of swim endurance capacity at two weeks. The regenerating fish gradually regained swim endurance at four and six weeks post-injury. In the swim behavior assay, controls swam steadily in the front part of the swim tunnel chamber corresponding to an elevated Y position.In contrast, at two weeks post-injury, injured fish could not maintain steady swim capacity against the current. Consequently, their swim tracks are more irregular, with an overall decrease in Y position. Y position increased at four and six weeks post-injury, indicating that regenerating animals gradually regained their ability to swim.Furthermore, relative to uninjured controls, lesioned animals at two weeks post-injury were markedly less active, stalled in the rear quadrant of the swim tunnel, and lost their ability to swim against low-current velocities. Consistent with their innate ability to achieve functional recovery, lesioned animals gradually normalized swim behavior parameters at four and six weeks post-injury. The use of the flow velocity control software in this protocol is optional.The alternative is to manually control the water current motor. The assays described in this study can be used to pre-screen for neural, muscular, or skeletal phenotypes. The tissue of interest can then be harvested for histological or molecular examination.Our lab and others have used this protocol to identify genes that are required for innate neural repair and factors that are sufficient to enhance neural regeneration.

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Neuroscience

2.9K ビュー.  Washington University School of Medicine. ゼブラフィッシュは、脊髄損傷から回復する能力のために貴重な神経再生モデルを提供します。この方法は、機能的な脊髄修復の読み出しとして水泳持久力および水泳行動を記述する。この技術は、成体ゼブラフィッシュの水泳持久力および水泳行動の信頼性および定量化可能な評価を提供する。これらの方法は、神経筋および筋骨格系の発達、疾患、および再生?...

ビデオ: Assessment of Swim Endurance and Swim Behavior 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...

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The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking...

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Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social...

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Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells,...

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dissection of the adult zebrafish kidney

Dissection of the Adult Zebrafish Kidney

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism.  Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

partial hepatectomy in adult zebrafish

Partial Hepatectomy in Adult Zebrafish

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver...

dissection of organs from the adult zebrafish

Dissection of Organs from the Adult Zebrafish

This protocol describes a procedure for identifying and dissecting organs from the adult...

intraperitoneal injection into adult zebrafish

Intraperitoneal Injection into Adult Zebrafish

We demonstrate intraperitoneal injection into adult zebrafish.  We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III.  This demonstration includes the use of cold water as an...

gavaging adult zebrafish

Gavaging 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...

in vivo surface electrocardiography for adult zebrafish

In Vivo Surface Electrocardiography for Adult Zebrafish

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult...

shuttle box assay as an associative learning tool for cognitive assessment in learning and memory studies using adult zebrafish

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

moderate prenatal alcohol exposure and quantification of social behavior in adult rats

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior  during adulthood.

a doxorubicin-induced cardiomyopathy model in adult zebrafish

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are...

transplantation of whole kidney marrow in adult zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

In this article, we demonstrate a method to perform HCT in adult...

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

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 ...

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

Modeling Amyloid-&amp;#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish.
Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Three-photon microscopy enables high-contrast fluorescence imaging deep in living biological tissues, such as mouse and zebrafish brains, with high spatiotemporal resolution.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo ...