We are grateful to the students in the Animal Behavior and Welfare Laboratory at Texas A&#38;M University for their assistance in decoding video recordings of cattle behavior and to the personnel at the Animal Science Teaching and Research Center for their assistance with this project. This project was supported by the Texas A&#38;M University Department of Animal Science Graduate Student Research Mini-Grant program.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Texas A&#38;M University (IACUC2016-0356).
1. Animal and housing
<ol>
	<li>House yearling &#188; Bos indicus x &#190; Bos taurus steers (n = 32) from the same herd in two drylot pens (n = 16 steers/pen) for 7 days prior to test commencement. At the beginning of the study, steers weighed 270.9 &#177; 14.8 kg and were fed the same standard growing ration throughout the study.</li>
	<li>Visually evaluate steers daily as part of routine husbandry practices. No steers received medical treatment throughout the duration of the study.</li>
</ol>
2. Description of the tests
<ol>
	<li>Test 1: Exit velocity
	<ol>
		<li>Place electronic timers in front of a handling chute so that the distance between the starting and stopping points is 1.8 m. These timers are designed to start timing when the animal breaks the first electronic beam and stop when the animal breaks the second electronic beam.</li>
		<li>Move cattle through the handling facility.</li>
		<li>Catch each animal in the headgate of the chute and keep it restrained for 10 s.</li>
		<li>After 10 s, release the animal from the headgate.</li>
		<li>With the electronic timer, record the time it takes for the animal to traverse 1.8 m from the chute.</li>
		<li>Calculate the velocity of the animal as it leaves the chute by dividing 1.8 m by the time it took for the animal to traverse the 1.8 m after release from the headgate and chute. 
		NOTE: Other publications have used this data collection strategy8,9,10,11.</li>
	</ol>
	</li>
	<li>Test 2: Chute score
	<ol>
		<li>Move the cattle through the handling facility.</li>
		<li>Catch each animal in the headgate of the chute for 10 s without applying pressure to its body.</li>
		<li>Have someone observe the cattle for the 10 s and assign each animal a score according to the 2019 Beef Improvement Federation Guidelines for Uniform Beef Improvement Programs 9th Edition (Table 2) based upon its behavior while being restrained.</li>
		<li>After 10 s, release animal from the headgate and chute. 
		NOTE: Other publications have used this data collection strategy12,13,14.</li>
	</ol>
	</li>
	<li>Test 3: Pen score
	<ol>
		<li>Place a group of five cattle in a pen (7.3 m W x 7.3 m L x 2.4 m H).</li>
		<li>Have a single human observer that is unknown to the cattle enter the pen on foot and close the gate after entering the pen.</li>
		<li>Have the observer take two steps towards the group of cattle.</li>
		<li>Visually observe each animal&#39;s behavior in response to the observer.</li>
		<li>Within 30 s of entering the pen, assign each animal a score according to the 2019 Beef Improvement Federation Guidelines for Uniform Beef Improvement Programs 9th Edition (Table 3).</li>
		<li>Clean the testing arena from urine and feces in between groups of animals. 
		NOTE: Other publications that have used this data collection strategy10,11.</li>
	</ol>
	</li>
	<li>Test 4: Bovine zero maze
	<ol>
		<li>Construct a Bovine Zero Maze (BZM).
		<ol>
			<li>Use cattle panels to create a circular track 1.6 m wide, with the inner and outer diameters measuring 6.6 m and 8.2 m, respectively (Figure 1).</li>
			<li>Divide the BZM into four quadrants of equal length with two opposing open quadrants and two opposing closed quadrants where the panels are covered with shade cloth and the shade cloth is stretched across the inner and outer rings of the maze to make a roof over the closed portions of the maze.</li>
			<li>If the test is conducted outdoors, to minimize variation due to shadows, orient the BZM such that the closed sections of the maze face north and south and conduct tests at approximately noon each test day.</li>
		</ol>
		</li>
		<li>Mount a video camera(s) to capture the entire arena. Turn the camera on and begin recording.</li>
		<li>Using low stress handling practices, move a single animal into an open portion of the maze, and allow the animal to explore the arena for 10 min.</li>
		<li>At the end of the 10 min observation period, return the animal to its home pen.</li>
		<li>Clean the testing arena from urine and feces in between animals.</li>
		<li>Decode the video recordings for frequency and latency of steps, escape attempts, kicks, urinations, defecations, vocalizations, standing bouts, duration of time spent standing, duration of time spent walking, latency to enter closed areas, number of times the animal enters closed areas, amount of time in closed/open portions, number of transitions between open/closed arms. Metrics were identified based upon previously published work15.</li>
	</ol>
	</li>
	<li>Test 5: Individual Startle Test and the Group Startle Test
	<ol>
		<li>Construct an arena (7.3 m W x 7.3 m L x 2.4 m H) that has a solid, uniform ground surface free of vegetation or manure, and two closed umbrellas at opposite ends of the arena (Figure 2). The umbrellas should be designed so that they open suddenly at the push of a button.
		<ol>
			<li>Ensure that the sides of the arena are solid or covered with plywood or shadecloth to ensure that the animal cannot see outside of the arena.</li>
			<li>Cut a hole at approximately cattle head height on opposite sides of the arena for the umbrella to penetrate through.</li>
		</ol>
		</li>
		<li>Mount a video camera(s) to capture the entire arena. Turn the video camera on and begin recording.</li>
		<li>Using low stress handling practices, move a single animal into the testing arena. For the group startle test introduce a small group of approximately four animals.</li>
		<li>After the animal(s) has been in the arena for 60 s, open the two umbrellas simultaneously.</li>
		<li>Leave the animal(s) in the arena for 4 min after the umbrellas have opened.</li>
		<li>Clean the testing arena from urine and feces in between tests.</li>
		<li>Decode the video recordings for the frequency and latency of steps, escape attempts, touching the umbrellas, kicks, urinations, defecations, vocalizations, standing bouts, duration of time spent standing, steps in the first 60 s of testing, and steps in the 60 s after the umbrellas were opened for each animal. Metrics were identified based upon previously published work16.</li>
	</ol>
	</li>
	<li>Test 6: Open field test
	<ol>
		<li>Construct a square arena (7.3 m W x 7.3 m L x 2.4 m H) that has a solid, uniform ground surface free of vegetation or manure. The sides of the arena should be solid or covered with plywood or shadecloth to ensure that the animal cannot see outside of the arena.</li>
		<li>Mount a video camera(s) to capture the entire arena. Turn the video camera(s) on and begin recording.</li>
		<li>Using low stress handling practices, move a single animal to the center of a solid sided open field testing arena.</li>
		<li>Leave the animal in the arena for 10 min.</li>
		<li>After 10 min, return the animal to its home pen.</li>
		<li>Clean the testing arena from urine and feces in between animals.</li>
		<li>Decode the video recordings for the frequency and latency to first step, escape attempts, kicks, urination, defecation, vocalization, standing bouts, duration of time spent standing, duration of walking, number of steps taken, number of steps taken during the first 60 s of testing. Metrics were identified based upon previously published work17,18,19.</li>
	</ol>
	</li>
</ol>
3. Statistical analysis
<ol>
	<li>Evaluate inter- and intra-test repeatability using a Pearson&#39;s Correlation (PROC CORR) and reliability calculated using Cronbach&#39;s alpha (PROC CORR). Conduct a validity of response variables with relation to average daily gain (ADG) using a regression analysis (PROC REG).</li>
	<li>After standardizing the variables (PROC STANDARD), use a Cluster Analysis (PROC VARCLUS) to identify relationships among variables from within and among different tests. Many of these variables may be regressed against production metrics to identify production-relevant relationships among cattle behavior during these tests and productivity.</li>
</ol>

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The authors have nothing to disclose.

Exit Velocity and Chute Score 
The EV and the CS are both evaluated while the animal is being processed through a handling chute. Although cattle behavior for both the EV and the CS are quantified during the same scenario, behavioral responses to these two tests are not related24. This suggests that the scenario in which the EV (e.g., escaping from restraint) and the CS (e.g., enduring restraint) are assessed may be perceived differently by cattle, and subsequently evaluate different emotional systems. The EV evaluates the behavior of cattle as they are escaping from restraint and is therefore thought to evaluate the FEAR system while the CS may evaluate RAGE. The CS evaluates the behavior of cattle while being restrained in the handling chute (Table 2), and thus may be a good proxy for the RAGE emotional system.
Substantial research has been conducted on the relationship between EV and production, health, and behavioral traits. While EV can be influenced by an animal&#39;s previous experience, this objective metric may be effective in quantifying the FEAR system, as substantial relationships between EV and health, productivity, breeding, and behavior have been identified. Cattle with faster EV have reduced growth rates14, poor carcass quality11,25, reduced immune function20, and higher cortisol levels during handling10. This measurement can provide information about behavior in the home pen, because EV is positively correlated with step counts in the home pen13. From an animal management perspective, cattle with faster EV are more difficult to handle, present greater risk to animal managers, and may influence the behavior of herd-mates. While EV may be a good metric for evaluating FEAR, it does not measure all emotional systems. Therefore, additional tests are required to evaluate all of the emotional systems influencing production and welfare.
Pen Score 
The PS subjectively evaluates the cattle&#39;s willingness to be approached by a human (Table 3) and may be useful in evaluating the PANIC system. However, the PS has been criticized for lack of objectivity, because different evaluators may have different interpretations of behavior, and several subjective evaluations have suffered from poor inter-rater reliability26.
Startle Test 
Anxiety is highly evolved in all prey species. High levels of FEAR help protect the animal from pain and activates the sympatho-adrenal and hypothalamic-pituitary-adrenal axes as part of the fight or flight and stress response to a perceived danger. The startle test evaluates an animal&#39;s response to sudden, novel stimuli, and has been identified as an effective measurement in identifying behavioral differences among different genetic strains of pigs27. The startle test may be effective in evaluating the sensitivity and reactivity of the sympatho-adrenal system, which has production-relevant consequences when activated and may provide insight into the FEAR system.
Open Field Test 
The OFT is the most commonly used test. The OFT was originally designed to evaluate individual animal boldness, or willingness to enter an open arena, an environment that may be perceived as dangerous and risky to the animal&#39;s survival. The OFT has been validated for species that instinctively seek shelter and avoid open spaces, such as rodents, chickens, and turkeys16.
Cattle evolved to live in open fields, thus the OFT may not induce the behavioral and physiological responses associated with FEAR and may be better suited to evaluate social isolation (PANIC/GRIEF) or exploration (SEEKING). Further, the OFT evaluates individual animals, and because cattle are gregarious herd animals, the experience of the OFT may be eliciting an emotional response other than FEAR. The OFT lacks a strong correlation with other FEAR tests and the results are difficult to interpret (i.e., many factors can lead to the same activity). Therefore, the OFT is not recommended as a general FEAR test for cattle16 and may not provide a comprehensive understanding of the FEAR systems in cattle. The OFT may, however, be a useful tool in quantifying either the PANIC or the SEEKING systems in cattle.
The SEEKING system is essential for animals to acquire the resources needed for survival. High SEEKING levels provoke intense, persistent enthusiastic exploration, appetitive and anticipatory excitement, and learning. This system can result in forward locomotion as the animal is motivated to explore its surroundings. SEEKING can play a role in both positive and negative emotions; positive SEEKING may engender a sense of purpose while negative SEEKING may result in behaviors associated with safety28. Cows that spent more time exploring and explored a larger portion of the range (e.g., stronger activation of SEEKING) ate quicker while in confinement, had calves with heavier weaning weights, higher cortisol concentrations during confinement, and shorter postpartum intervals to estrus29. Therefore, the SEEKING system can have production and welfare implications. Identifying animals with high activation of the SEEKING system may be more successful in extensive ranging environments where individual and reproductive fitness is dependent on the animal&#39;s capacity to find resources and shelter. However, animals with high activation of the SEEKING system may experience higher levels of stress and frustration during confinement.
Bovine Zero Maze 
Commonly used tests in biomedical research that are designed to evaluate the efficacy of anti-anxiety and anti-depressant drug development in rodents are the elevated plus maze (EPM) and the elevated zero maze (EZM)30. These tests exploit the instinctual behavior of the rodent and its natural propensity for dark, closed-in places to quantify their willingness to explore environments that would be inherently fearful or induce anxiety. Metrics from these tests can include the latency to leave the darkened arm of the maze, duration of time in the open and closed arms of the maze, and the number of transitions between the two environments during the testing period as well as the behavior of the animal (e.g., vocalization, urination, defecation, escape attempts) during the test31.
The EPM and the EZM are both well validated tests for quantifying FEAR/ANXIETY in rodents15,31. A modified EPM has been used to quantify the FEAR response in swine32 but has not been utilized in ruminants. However, the EPM has been criticized for its ambiguity of interpretation regarding behavior in the central square of the maze. Therefore, the EZM was designed to evaluate the same metrics as the EPM but allows uninterrupted exploration without ambiguity. When identifying a test to evaluate FEAR/ANXIETY and SEEKING in cattle, the EZM was a logical model. The EZM is conducive to the natural behavior of cattle, as they instinctively move in circular patterns and have a propensity for returning to the areas from which they came.
By applying principles similar to the EZM with an inverse of interpretation, a Bovine Zero Maze33 has been developed to evaluate the FEAR, PANIC/GRIEF, and SEEKING systems in cattle. Cattle evolved to live in open spaces; therefore, cattle with reduced activation of the FEAR and PANIC/GRIEF systems will be more willing to spend time in the open portions of the BZM than the darkened portions of the maze, will be less likely to enter the closed portions of the maze, and will perform more escape attempts.
Quantifying cattle behavior across multiple evaluations may identify complex emotional relationships that can have economic significance, are easily measured, and can be included in breeding selection efforts. The emotional circuits of PLAY, LUST, and CARE were not evaluated in this study.

Animal temperament has been linked to behavioral characteristics such as exploratory behavior and boldness1,2 and can exhibit consistencies over time and across contexts3,4. However, temperament is composed of multiple emotional systems working together. Animals experience physical and psychological stressors, and evaluating the emotional response to both types is challenging. Emotional state can influence how animals perceive stimuli (e.g., cognitive bias), and is a critical component of animal welfare5. Understanding how an individual will behave in response to psychological stressors (e.g., commingling, weaning, change in stockperson) will provide animal managers additional selection criteria when identifying animals that have the skills to cope with psychological stressors.
Emotions are controlled by seven core affective systems within the brain (Table 1)6. These systems include four that control positive emotions: 1) SEEKING (exploration), 2) LUST (sexual excitement), 3) CARE (nurturance), and 4) PLAY (social joy). Three systems control negative emotions 1) FEAR (anxiety), 2) RAGE (anger), and 3) PANIC/GRIEF (separation distress). These affective systems may be heritable7, impact profitability, and are a critical component of animal welfare.
A battery of tests has been developed to evaluate cattle temperament (e.g., exit velocity, chute score). However, the evaluation of temperament has the potential to be influenced by current conditions, previous experiences, and observer bias. While many of these behavioral evaluations are commonly referred to as fear tests, they may be quantifying different emotional components of temperament other than FEAR. In addition, the variation in how these tests have been conducted makes comparisons across different evaluations challenging. Thus, there is a need to understand the relationships among these behavioral evaluations as well as have a standardized protocol for these temperament evaluations.
The goal of this article is to visually document the different fear tests used for cattle; present the type of data that were generated from these different tests; evaluate the repeatability, validity, and reliability of these tests; demonstrate how to evaluate the relationships among the behaviors captured from these tests; and suggest which emotional circuit could be evaluated with each test.

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the use of traditional fear tests to evaluate different emotional circuits in cattle

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli may facilitate breeding selections and identify animals that are better suited to specific management strategies. Multiple tests have been developed to evaluate cattle temperament (e.g., exit velocity, chute score, pen score, open field test, startle test, bovine zero maze), but each of these tests evaluates the animal&#39;s response to different stimuli (e.g., isolation, novel environment, startle, willingness to enter an enclosed area). Cattle temperament has been observed to be relatively stable over time. However, the evaluation of temperament has the potential to be influenced by current conditions, previous experiences, and observer bias. Many of these temperament tests have been improperly categorized as fear tests and have also been criticized for being subjective. This paper provides a framework for standardizing behavioral tests for cattle and suggests that these different evaluations assess different aspects of the animal&#39;s overall temperament.

Watch this Scientific Journal Video about The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle at JoVE.com

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

Here, we present a protocol to conduct a variety of behavioral tests in cattle that have been designed to evaluate emotions. A battery of behavioral tests (open field test, startle test, bovine zero maze, exit velocity, pen score, and chute score) were conducted to evaluate different components of animal temperament.

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli ...

the-use-traditional-fear-tests-to-evaluate-different-emotional

Universidad San Sebastian

Research

JoVE Journal

Behavior

8.5K Views.  Texas A&M University. Oké, we hebben besproken hoe deze verschillende scoresystemen verschillende gegevens krijgen, en je hebt hele mooie statistieken en clusteranalyse gedaan om te laten zien hoe dit werkt, en het zou echt goed zijn om het uit te leggen voor iedereen die kijkt. Zeker. Over hoe dit werkt en leg het op een toegankelijke manier uit. Ja, we hebben al deze verschillende gedragsreacties van deze dieren gemeten in verschillende scenario's en, die ontworpen waren om specifieke emoties uit te lokken, dus...

Video: The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., &#8220;startle probes&#8221;). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.

Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science.

Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Medicine

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 ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...

Assessing Rodent Empathy: Observational Fear and Vicarious Freezing Method

Observational Fear as a Model of Affective Empathy in Mice

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows individuals to respond to the emotional states of others, promoting prosocial behaviors and social bonding. Observational fear is a fundamental aspect of affective empathy, where an observer witnesses a demonstrator undergoing aversive experiences and subsequently exhibits fear behaviors. This socially induced vicarious freezing response in observers, known as emotional contagion or affect sharing, is regarded as an indicator of empathy-like traits in rodents. Here, we present a protocol that delineates the assessment of vicarious freezing responses in observers exposed to conspecific demonstrators receiving aversive foot shocks. The utilization of observational fear assays in rodents has become a widely adopted method for studying the neural mechanisms underlying affective empathy. Given the universality of observational fear across mammals, this methodology further contributes to advancing our understanding of the neural substrates of empathy in humans.

The observational fear paradigm assesses vicarious freezing in rodents as a model for affective empathy. This procedure entails exposing observer rodents to conspecific demonstrators receiving aversive foot shocks to elicit empathic freezing responses. By employing observational fear assays, researchers can investigate the neural mechanisms underlying affective empathy.

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows ...

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

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