Name: boldness, aggression, and shoaling assays for zebrafish behavioral syndromes
Uploaded: 2016-08-29T13:00:00
Description: Watch this Scientific Journal Video about Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes at JoVE.com

This work was supported by a Howard Hughes Medical Institute Education Grant and an internal grant from the Saint Joseph's University chapter of Sigma Xi. We would also like to thank the three anonymous reviewers who helped strengthen the protocol and interpretations.

The following methodologies for the housing, care, and study of zebrafish have been approved by the Saint Joseph&#39;s University IACUC.
1. Zebrafish Housing and Care
<ol>
	<li>Obtain the subject zebrafish from a local supplier, or from wild-caught populations. Please note that housing the zebrafish is subject to IACUC guidelines and additional permits are required for housing wild-caught populations.</li>
	<li>Identify and separate zebrafish according to sex. Alternatively, skip this step o...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggression+and+vasotocin+are+associated+with+dominant-subordinate+relationships+in+zebrafish.">Aggression and vasotocin are associated with dominant-subordinate relationships in zebrafish.</a> Behav Brain Res. 167 (1), 94-102 (2006).</li><li>McRobert, S., Bradner, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+body+coloration+on+shoaling+preferences+in+fish.">The influence of body coloration on shoaling preferences in fish.</a> Anim Behav. 56 (3), 611-615 (1998).</li><li>Ruhl, N., McRobert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sex+and+shoal+size+on+shoaling+behaviour+in+Danio+rerio.">The effect of sex and shoal size on shoaling behaviour in Danio rerio.</a> J Fish Biol. 67 (5), 1318-1326 (2005).</li><li>Snekser, J., Ruhl, N., Bauer, K., McRobert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+sex+and+phenotype+on+shoaling+decisions+in+zebrafish.">The influence of sex and phenotype on shoaling decisions in zebrafish.</a> J Comp Psychol. 23, 70-81 (2010).</li><li>Budaev, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Principal+Components+and+Factor+Analysis+in+Animal+Behaviour+Research:+Caveats+and+Guidelines.">Using Principal Components and Factor Analysis in Animal Behaviour Research: Caveats and Guidelines.</a> Ethology. 116 (5), 472-480 (2010).</li><li>Paciorek, T., McRobert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daily+variation+in+the+shoaling+behavior+of+zebrafish+Danio+rerio.">Daily variation in the shoaling behavior of zebrafish Danio rerio.</a> Curr Zool. 58 (1), 129-137 (2012).</li><li>Wright, D., Krause, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+measures+of+shoaling+tendency+in+zebrafish+(Danio+rerio)+and+other+small+teleost+fishes.">Repeated measures of shoaling tendency in zebrafish (Danio rerio) and other small teleost fishes.</a> Nat Protoc. 1 (4), 1828-1831 (2006).</li><li>Cachat, J., Stewart, 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=Measuring+behavioral+and+endocrine+responses+to+novelty+stress+in+adult+zebrafish.">Measuring behavioral and endocrine responses to novelty stress in adult zebrafish.</a> Nat Protoc. 5 (11), 1786-1799 (2010).</li><li>Croft, D. 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A., Wedderburn, R. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+Linear+Models.">Generalized Linear Models.</a> J Roy Stat Soc A Sta. 135, 370-384 (1972).</li><li>Iman, R. L., Conover, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distribution-free+approach+to+inducing+rank+correlation+among+input+variables.">A distribution-free approach to inducing rank correlation among input variables.</a> Commun Stat Simulat. 11 (3), 311-334 (1982).</li><li>Fleiss, J. L., Cohen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Equivalence+of+Weighted+Kappa+and+the+Intraclass+Correlation+Coefficient+as+Measures+of+Reliability.">The Equivalence of Weighted Kappa and the Intraclass Correlation Coefficient as Measures of Reliability.</a> Educ Psychol Meas. 33 (3), 613-619 (1973).</li><li>Ruhl, N., McRobert, S. P., Currie, W. J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoaling+preferences+and+the+effects+of+sex+ratio+on+spawning+and+aggression+in+small+laboratory+populations+of+zebrafish+(Danio+rerio).">Shoaling preferences and the effects of sex ratio on spawning and aggression in small laboratory populations of zebrafish (Danio rerio).</a> Lab Animal. 38 (8), 264-269 (2009).</li><li>Bell, A. 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The protocol will determine if there are consistent associations in boldness, aggression, and shoaling behaviors in zebrafish. If there are consistent associations in a given population between any of these behaviors, then a behavioral syndrome is present. By studying a population&#39;s natural behavioral syndrome, researchers can have a more complete understanding of its behavioral dynamic, population structure, and possibly evolutionary history3. Furthermore, manipulating the environment that affects these b...

There is a growing body of literature investigating the associations between distinct behaviors within individual animals from a given population. These associations are termed behavioral syndromes, and the measurements typically include boldness, aggression, exploratory behavior, and sociability1-5. Behavioral syndromes are valuable for both direct and indirect reasons. Directly, knowledge of behavioral syndromes can provide a more complete view of evolutionary theory, population structure, and population dynamics3. Indirectly, knowledge regarding behavioral associations may inform fields that quantify behavior such as pharmacology6 , toxicology7, behavioral genetics8,9, and endocrinology10. Because of these direct and indirect benefits, an increased knowledge of behavioral syndromes is especially valuable in commonly used model organisms such as the zebrafish. Studies using zebrafish are found in a variety of disciplines, including the analysis of behavioral syndromes11-13. To advance knowledge in behavioral syndrome research, and because other disciplines also use behavioral measurements for hypothesis testing, reliable and succinct descriptions of behavior are required for valid analyses and interpretations and standardized protocols will facilitate inter-study comparisons within species. Our protocol was developed to measure a boldness-aggression-shoaling behavioral syndrome in a population of lab-reared zebrafish14. However, the basis of the protocol (tracking individual fish, ensuring proper randomization, and appropriate analyses) can be easily modified for a variety of alternative behavioral measures. Additionally, boldness, aggression, or shoaling assays can be run individually for the testing of distinct hypotheses. Therefore, while it is our goal to describe how to conduct a behavioral syndrome study and the protocol for successful individual level behavioral measurement, each facet of this procedure can stand alone.
The literature on behavioral syndromes spans several taxonomic groups, from arthropods to humans4 and, in order to measure a behavioral syndrome, at least two behavioral contexts must be quantified. Unfortunately, there is often little consistency in the assays that are used to quantify the behavioral measurement across the axes of behavior. For example, in fish, boldness may be measured using T-maze assays, open-field assays, or introduction of a novel or foreign stimulus15. Aggression studies in fish might involve dyad interactions, video stimulus assays, or clay model assays12,16,17. Likewise, analysis of shoaling behavior, which typically involves the measurement of shoalmate preference, may be performed in different types of tanks, with different methods to determine association time21-23. In this protocol a specific subset of the overall behavioral assay repertoire is presented. Specifically, this protocol presents a methodology to track individuals through boldness, aggression, and shoaling assays in a way that facilitates comparisons within individuals to determine whether the comparisons are consistent across all individuals within a population. We have performed this protocol with zebrafish and convict cichlids (Amatitlania nigrofasciata) in previous studies14,18, and it will work with any similarly sized freshwater fish.
Boldness assays are conducted in a 1.5 L trapezoidal tank that has a horizontal line delineating equal sized areas in the upper and lower portions of the tank. Quantified behaviors include the number of transitions by the test fish between the upper and lower portion of the tank, the time spent in each portion, the number of darts, and the latency to enter the upper portion. The aggression assay is performed in a 19L rectangular tank that includes a 3 inch x 5 inch mirror inclined at about 22&#176; situated in the lower left corner of the tank19. Quantified behaviors include the total amount of time spent by the target fish interacting with the mirror20, along with specific aggressive indicators &#8211; number of bites, lateral displays, darts between the test fish and its reflection. For these specific indicators, bites are defined as quick lunges toward the mirror with an open mouth, lateral displays are defined as the flaring of lateral, pectoral, anal and dorsal fins in the direction of the mirror, and darts are any erratic movements that are not directed toward the mirror. Lastly, the shoaling assay quantifies the behavior of a test fish in center chamber of a tri-chambered tank. The side chambers of the tank are either empty, or contain a &#34;target shoal&#34; of fish, and the time the test fish spends near each side chamber is measured21-23. A single composite score, referred to as Strength of Shoaling (SoS), is calculated for each individual test fish, specific to the stimuli, and can be used in downstream analyses14. All behaviors are scored by a single viewer, or multiple viewers using free behavioral quantification software known as JWatcher24.
Testing the presence of a behavioral syndrome is primarily a statistical endeavor, and it is advisable to follow the guidelines as presented by Budeav 201025. Specifically, it is recommended to perform a principal components analysis (PCA) on centered and normed data in which the inputs are the vectors of an individual&#39;s behaviors in assays with multiple behavioral measurements (i.e., boldness and aggression). The PCA, performed on a correlation matrix, reduces the dimensionality of the behavioral measurements, and thus extracts the most important knowledge that explains a majority of the variation. The extracted components can then be interpreted based on high factor loadings for the individual behavior of interest and regression scores can be extracted for each individual on the basis of the explanatory components. These regression scores can then be compared to the SoS measurement and other various non-behavioral measurements such as fish size or sex.
This workflow has been implemented in a study of zebrafish behavioral syndromes in which a sex specific behavioral syndrome that exists between boldness and shoaling14 was discovered. In this situation, bolder zebrafish males are more likely to associate with a larger, more aggressive species (D. albolineatus), but this association is lost in females. This workflow was also implemented in a study of juvenile convict cichlid kin (Amatitlania nigrofasciata)18 in which a behavioral syndrome was not discovered, potentially indicating behavioral plasticity of the species. Therefore, the following protocol is presented with a goal of delineating the nature of three specific assays (boldness, aggression, and shoaling) in the framework of studying an individual level behavioral syndrome.

<table border="0" cellpadding="0" cellspacing="0" width="783">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Zebrafish Rack System</td>
			<td style="width:224px;">Aquaneering Inc</td>
			<td style="width:119px;">Cat. # ZS550</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pet Valu Tropical Fish Food, 224.0g</td>
			<td>Pet Valu</td>
			<td>Cat. # 31700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Brine Shrimp Eggs, 16 oz</td>
			<td>Brine Shrimp Direct</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1.5 L Trapezoidal Tank</td>
			<td>Pentair Aquatic Ecosystems</td>
			<td>Cat. # itsts-a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">19L rectangular tank</td>
			<td>That Fish Place</td>
			<td align="right">211932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">76L rectangular tank</td>
			<td>That Fish Place</td>
			<td align="right">212180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hitachi KP-D20A CCD Camera</td>
			<td>Prescott&#39;s, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon AF Nikkor 35-105mm f/305~4.5s MACRO lens</td>
			<td>Nikon Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ArtMinds Square Mirror, Value Pack 3&#34;x3&#34;</td>
			<td>Michaels</td>
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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.

Depending on the nature of the study, and specific protocol employed, several distinct results are possible in a behavioral syndromes experiment. The following tables and figures, where indicated, are adapted from our previous study published in the journal Behavioural Processes14 and the journal Zebrafish17. When the proposal (as described above) is carried out in its entirety, two sets of results, &#39;within assay correlations&#39; and &#39;between assay correlati...

Watch this Scientific Journal Video about Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes at JoVE.com

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

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 ...

The overall goal of this protocol is to standardized the methodology used to observe, analyze and interpret boldness, aggression, shoaling behavioral syndromes in Zebrafish. Directly, this method can answer questions in evolutionary theory, population structure and population dynamics. When combined with interdisciplinary techniques it can be applied to fields such as pharmacology, toxicology, behavioral genetics and endocrinology.The main advantage of this technique is that the boldness, aggression, shoaling combination encompasses many well studied, easily quantifiable behaviors in Zebrafish. And each assay can be modified to answer alternative experimental questions. Begin by randomly separating the Zebrafish into the appropriate ratio of focal fish to stimulus fish.Store all fish in Zebrafish housing racks under a strict 12 to 12 hour light/dark cycle. Feed flake food to all focal and stimulus fish everyday. And only feed brine shrimp once a week on non-research days.Next, set up three assay tanks in the laboratory. One for aggression, boldness and shoaling in locations that minimize the possibility for adverse external stimuli. And allow enough room for a video camera to capture the entire tank.Set up the video camera, so that it has enough clarity to view the tank labels, as well as to be to distinguish subtle behaviors of the focal fish. Keep the camera maintained in the same position with a full view of the tank during all behavioral assay recordings, in order to minimize unintended differences in behavioral scoring as a result of differential recordings. Next, define a labeling scheme to track the date, fish number and assay number for all behavioral assays.Label the outside of the tank with tape in view of the camera. Record this information in a lab notebook for quick reference and future quality control. Finally, set up an acclimation tank that will be used between all assays, as well as directly after the individuals are removed from the housing rack.Begin by obtaining a 19 liter rectangular tank, measuring 30 by 15 by 10 centimeters. Outfit the tank with a external opaque partitions surrounding three sides of the aggression tank, leaving the front exposed for viewing to reduce undesirable outside stimuli. Divide the tank with external markings into four equal rectangular quadrants.Use silicone caulk to fix a mirror within the left lower quadrant, inclined at 22.5 degrees, forming a right triangle against the far left side of the tank. Have fresh, aged water, pre-warmed to 27.5 to 28.5 degrees celsius, ready to add to the aggression tank in order to ensure that the temperature of the water in the acclimation tank and aggression tank are within two degrees celsius of the housing rack. Cup a focal fish from the housing rack and transfer it into a separate acclimation tank where it will acclimate for 10 minutes.Confirm that the video camera is focused on the aggression assay tank and press record. Then, immediately transfer the focal fish via cupping from acclimation tank to the aggression tank. Record the aggression tank for 10 minutes and 30 seconds.Then, cup the focal fish from the aggression tank back into the acclimation tank. When watching the recording after the assay is complete, wait 30 seconds after the fish has been introduced to the tank as an additional acclimation period before scoring behaviors. Finally, score a single aggression assay at least twice until the assay is scored with high agreement between iterations.To set up the boldness assay, first obtain a small 1.5 liter trapezoidal tank, measuring 15 centimeters tall with a 26.5 centimeter top and 22.5 centimeter bottom, and a 6 centimeter width. Wrap opaque paper around three sides of the tank, and leave the front exposed for viewing. Place a single thin piece of tape external to the tank to demarcate the top from the bottom portion of the tank.Next, ensure that temperature of the water in the boldness tank is within two degrees celsius of the housing rack. Transfer the focal fish from the housing rack to the acclimation tank and let it acclimate for 10 minutes. Ensure that the video camera is focused on the boldness assay and press record on the device.Immediately transfer the focal fish from the acclimation tank to the boldness tank and record for eight minutes and 30 seconds. When watching the recording after the assay is complete, allow for an additional 30 second acclimation period after the fish is introduced to the tank before scoring. Finally, score a single boldness assay at least twice, until the assay is scored with high agreement between iterations.To set up the shoaling assay, obtain a 76 liter tank that has three compartments divided by two glass dividers sealed with silicone caulk. Externally, use tape to mark two preference zones that are six point three five centimeters from each of the glass dividers that designate each stimulus zone. Apply external opaque partitions around three sides of the shoaling tank and leave the front exposed for viewing.Next, select stimulus shoals to acclimate for at least 12 hours before testing and have them remain in the shoaling tank throughout the course of the experiment. Transfer the focal fish from the housing rack into a separate acclimation tank via cupping where it will acclimate for 10 minutes. Then transfer, by cupping, the focal fish from the acclimation tank to the central compartment of the shoaling tank.Allow for a 10 minute acclimation period before a 10 minute live scoring observation period, in which shoaling fish are defined as being within the preference zone in the chamber housing the target shoal. Next, quantify the time spend in each preference zone, as well as the time spent in the central volume. Finally, after the assays are completed, transfer the fish into a separate terminal tank that will serve as the final destination tank for all used focal individuals.This protocol looked at consistent associations in aggression, boldness and shoaling behaviors of Zebrafish to determine if a behavioral syndrome was present. Here, component one represents aggression behaviors, while component two is associated with boldness behaviors. Since aggression and boldness behaviors are not represented by the same component, it can be concluded that there is an absence of boldness aggression behavioral syndrome in Zebrafish.Once mastered, the entire protocol of one focal fish can be done in 70 minutes. While attempting this procedure, it's important to remember to familiarize yourself and your team with each target behavior in order to minimize observer bias. Scoring the same video recording multiple times will also increase confidence in your results.Following this procedure, other methods in pharmacological, genetic or physiological fields can be performed in order to answer additional questions about what internal and external factors affect behavioral syndromes. After watching this video, you should have a good understanding of how to observe, analyze and interpret boldness, aggression, shoaling behavioral syndromes in Zebrafish. Thanks for watching and good luck with your experiments!

boldness-aggression-shoaling-assays-for-zebrafish-behavioral

Research

JoVE Journal

Medicine

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Eye Tracking Young Children with Autism

The rise of accessible commercial eye-tracking systems has fueled a rapid increase in their use in psychological and psychiatric research. By providing a direct, detailed and objective measure of gaze behavior, eye-tracking has become a valuable tool for examining abnormal perceptual strategies in clinical populations and has been used to identify disorder-specific characteristics1, promote early identification2, and inform treatment3. In particular, investigators of autism spectrum disorders (ASD) have benefited from integrating eye-tracking into their research paradigms4-7. Eye-tracking has largely been used in these studies to reveal mechanisms underlying impaired task performance8 and abnormal brain functioning9, particularly during the processing of social information1,10-11. While older children and adults with ASD comprise the preponderance of research in this area, eye-tracking may be especially useful for studying young children with the disorder as it offers a non-invasive tool for assessing and quantifying early-emerging developmental abnormalities2,12-13. Implementing eye-tracking with young children with ASD, however, is associated with a number of unique challenges, including issues with compliant behavior resulting from specific task demands and disorder-related psychosocial considerations. In this protocol, we detail methodological considerations for optimizing research design, data acquisition and psychometric analysis while eye-tracking young children with ASD. The provided recommendations are also designed to be more broadly applicable for eye-tracking children with other developmental disabilities. By offering guidelines for best practices in these areas based upon lessons derived from our own work, we hope to help other investigators make sound research design and analysis choices while avoiding common pitfalls that can compromise data acquisition while eye-tracking young children with ASD or other developmental difficulties.

Eye tracking has long been used to study gaze patterns in typically-developing individuals, but recent technological advancements have made its use with clinical populations, including autism, more feasible. While eye-tracking young children with autism can offer insight into early symptom manifestations, it involves methodological challenges. Suggestions for best practices are provided.

The rise of accessible commercial eye-tracking systems has fueled a rapid increase in their use in psychological and psychiatric research. By providing a ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user&#8217;s specific biological and technical needs.

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...