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

<ol>
	<li>Becker, C. G., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regeneration+from+ependymo-radial+glial+cells:+cook,+little+pot,+cook.">Neuronal regeneration from ependymo-radial glial cells: cook, little pot, cook.</a> Developmental Cell. 32 (4), 516-527 (2015).</li><li>Mokalled, M. H., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regeneration+toolkit.">A regeneration toolkit.</a> Developmental Cell. 47 (3), 267-280 (2018).</li><li>Orger, M. B., de Polavieja, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+behavior:+opportunities+and+challenges.">Zebrafish behavior: opportunities and challenges.</a> Annual Review of Neuroscience. 40, 125-147 (2017).</li><li>Becker, C. G., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+zebrafish+as+a+model+for+successful+central+nervous+system+regeneration.">Adult zebrafish as a model for successful central nervous system regeneration.</a> Restorative Neurology and Neuroscience. 26 (2-3), 71-80 (2008).</li><li>Gurevich, D. B., 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=Asymmetric+division+of+clonal+muscle+stem+cells+coordinates+muscle+regeneration+in+vivo.">Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo.</a> Science. 353 (6295), (2016).</li><li>Mokalled, M. H., 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=Injury-induced+ctgfa+directs+glial+bridging+and+spinal+cord+regeneration+in+zebrafish.">Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish.</a> Science. 354 (6312), 630-634 (2016).</li><li>Kizil, C., Kaslin, J., Kroehne, V., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+and+brain+regeneration+in+zebrafish.">Adult neurogenesis and brain regeneration in zebrafish.</a> Developmental Neurobiology. 72 (3), 429-461 (2012).</li><li>Wolman, M. A., 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+genome-wide+screen+identifies+PAPP-AA-mediated+IGFR+signaling+as+a+novel+regulator+of+habituation+learning.">A genome-wide screen identifies PAPP-AA-mediated IGFR signaling as a novel regulator of habituation learning.</a> Neuron. 85 (6), 1200-1211 (2015).</li><li>Granato, 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=Genes+controlling+and+mediating+locomotion+behavior+of+the+zebrafish+embryo+and+larva.">Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva.</a> Development. 123, 399-413 (1996).</li><li>Brockerhoff, S. E., 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+behavioral+screen+for+isolating+zebrafish+mutants+with+visual+system+defects.">A behavioral screen for isolating zebrafish mutants with visual system defects.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (23), 10545-10549 (1995).</li><li>Moens, C. B., Yan, Y. L., Appel, B., Force, A. G., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valentino:+a+zebrafish+gene+required+for+normal+hindbrain+segmentation.">Valentino: a zebrafish gene required for normal hindbrain segmentation.</a> Development. 122 (12), 3981-3990 (1996).</li><li>Cavone, L., 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+unique+macrophage+subpopulation+signals+directly+to+progenitor+cells+to+promote+regenerative+neurogenesis+in+the+zebrafish+spinal+cord.">A unique macrophage subpopulation signals directly to progenitor cells to promote regenerative neurogenesis in the zebrafish spinal cord.</a> Developmental Cell. 56 (11), 1617-1630 (2021).</li><li>Reimer, M. 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=Motor+neuron+regeneration+in+adult+zebrafish.">Motor neuron regeneration in adult zebrafish.</a> Journal of Neuroscience. 28 (34), 8510-8516 (2008).</li><li>Klatt Shaw, D., 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=Localized+EMT+reprograms+glial+progenitors+to+promote+spinal+cord+repair.">Localized EMT reprograms glial progenitors to promote spinal cord repair.</a> Developmental Cell. 56 (5), 613-626 (2021).</li><li>Ahmed, R. U., Alam, M., Zheng, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+spinal+cord+injury+and+behavioral+tests+in+laboratory+rats.">Experimental spinal cord injury and behavioral tests in laboratory rats.</a> Heliyon. 5 (3), 01324 (2019).</li><li>Pajoohesh-Ganji, A., Byrnes, K. R., Fatemi, G., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+scoring+method+to+assess+behavioral+recovery+after+mouse+spinal+cord+injury.">A combined scoring method to assess behavioral recovery after mouse spinal cord injury.</a> Neuroscience Research. 67 (2), 117-125 (2010).</li><li>Basso, D. M., Beattie, M. S., Bresnahan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+reliable+locomotor+rating+scale+for+open+field+testing+in+rats.">A sensitive and reliable locomotor rating scale for open field testing in rats.</a> Journal of Neurotrauma. 12 (1), 1-21 (1995).</li><li>Scheff, S. W., Saucier, D. A., Cain, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+statistical+method+for+analyzing+rating+scale+data:+the+BBB+locomotor+score.">A statistical method for analyzing rating scale data: the BBB locomotor score.</a> Journal of Neurotrauma. 19 (10), 1251-1260 (2002).</li><li>Li, Q., 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=Differential+behavioral+responses+of+zebrafish+larvae+to+yohimbine+treatment.">Differential behavioral responses of zebrafish larvae to yohimbine treatment.</a> Psychopharmacology (Berl). 232 (1), 197-208 (2015).</li><li>Wakamatsu, Y., Ogino, K., Hirata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+capability+of+zebrafish+is+governed+by+water+temperature,+caudal+fin+length+and+genetic+background.">Swimming capability of zebrafish is governed by water temperature, caudal fin length and genetic background.</a> Scientific Reports. 9 (1), 16307 (2019).</li><li>Ahmed, O., Seguin, D., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+predator+avoidance+task+in+zebrafish.">An automated predator avoidance task in zebrafish.</a> Behavioral Brain Research. 216 (1), 166-171 (2011).</li><li>Conradsen, C., McGuigan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexually+dimorphic+morphology+and+swimming+performance+relationships+in+wild-type+zebrafish+Danio+rerio.">Sexually dimorphic morphology and swimming performance relationships in wild-type zebrafish Danio rerio.</a> Journal of Fish Biology. 87 (5), 1219-1233 (2015).</li><li>Leris, I., Sfakianakis, D. G., Kentouri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+zebrafish+Danio+rerio+males+better+swimmers+than+females.">Are zebrafish Danio rerio males better swimmers than females.</a> Journal of Fish Biology. 83 (5), 1381-1386 (2013).</li></ol>

The authors have no conflicts of interest.

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. ...

assessment-of-swim-endurance-and-swim-behavior-in-adult-zebrafish

Research

JoVE Journal

Neuroscience

3.1K Views.  Washington University School of Medicine. 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.

Video: Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

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 Mouse Forced Swim Test

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 ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

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 ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

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.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

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.

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 ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

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.

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Studying the Integration of Adult-born Neurons

The Mouse Forced Swim Test

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

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

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

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