Mohammed R. Milad is supported by NIMH grant R01 MH097880 and institutional funds from the Department of Psychiatry at MGH.

NOTE: All procedures in this protocol have been approved by the Subcommittee on Research Animal Care, which serves as the Institutional Animal Care and Use Committee (IACUC) for the Massachusetts General Hospital, and are in compliance with the National Institutes of Health guidelines.
1. Animal Housing and Handling Procedure
<ol>
	<li>Upon arrival, house adult Sprague Dawley female rats about 8-10 weeks of age (weighing 200-225 g) in groups of 3-4 inch plastic bins with woodchip bedding. Maintain them on a diet of ad libitum access to chow and water and a 12 hr light/dark cycle. Leave rats to acclimate to the housing conditions and animal facility for a minimum of 1 week. 
	NOTE: All procedures in this manuscript are conducted during the light cycle.</li>
	<li>Following this acclimation period, handle all animals for 5 min each for 2-3 days. Properly pick up the rats one at a time (supported by the torso and not by the tail), hold them securely, and pet them during this time. Handle animals that appear more stressed, jumpy, or anxious for longer periods of time. Do this to minimize handling stress, which could affect the following steps in this protocol. 
	NOTE: The number of days and amount of time handling the animals can vary between different labs; however, these should be consistent between experiments within the same lab.</li>
</ol>
2. Monitoring the Estrous Cycle: Vaginal Swab Smearing and Staining Procedure
<ol>
	<li>Make 0.9% saline solution (sodium chloride dissolved in distilled water). Moisten a cotton-tipped applicator in this solution. Blot the tip using a paper towel to avoid oversaturation, which could lead to inadequate sample collection or loss of cells. Holding the tail upward away from the vaginal opening, gently insert the cotton tip into the vaginal canal and roll around the walls to capture loose vaginal cells.</li>
	<li>If the animal urinates on the cotton tip, replace with a new one, as urine contamination will make it difficult for phase identification. Be extremely careful as to not cause distress to the animal during this procedure because exposure to stress will disrupt the estrous cycle. When swabbing, aim to achieve a swift insertion and withdrawal that is not too deep as to minimize extra stimulation to the cervix, which can induce pseudopregnancy. 
	NOTE: Pseudopregnancy can be identified as an acyclic estrous cycle that typically lasts for about 12 days, i.e. consecutive days of diestrus25.</li>
	<li>Carefully remove the applicator tip and roll it onto a pre-labeled microscope slide. Avoid pressing down too hard as too much fluid on the slide can make phase identification difficult once dry. 
	NOTE: One slide can contain multiple days for each animal, although this can be done according to individual preferences.</li>
	<li>Make sure the sample transfers onto the slide. Collect samples for several consecutive days on one slide to be able to better track and identify daily estrous phases changes. 
	NOTE: If possible, it is also ideal if the people collecting samples remain the same for all animals.</li>
	<li>Once the slide is dry, begin the staining protocol for phase identification. Using the staining kit (see Table of Materials), immerse slides 10 times in the fixative (light blue), 10 times in stain solution (pink), and 5 times in counterstain solution (purple). Rinse slides gently under running water. Let slides completely air dry before viewing under microscope. 
	NOTE: The typically 4-5 day long estrous cycle consists of (in this order): estrus (E), metestrus (M), diestrus (D), and proestrus (P)26. Estrus is characterized by blue, cornified cells, metestrus by leukocytes and a combination of cornified and nucleated cells, diestrus is similar to metestrus in presence of cell types except they are very sparse, and proestrus by purple-stained aggregates of nucleated cells (Figure 1). There are various staining procedures, in addition to those described above, that are equally effective for vaginal smear cytology.</li>
	<li>Collect samples for at least two complete cycles (~8-10 days) in order to monitor the phases accurately prior to experimentation. Be consistent with the time of day the samples are taken. Collect vaginal smear samples daily throughout and until the end of the experiment.</li>
</ol>
3. Pre-exposure
<ol>
	<li>Prior to the initiation of the experiment, pre-expose the animals to the conditioning chambers allow the animals to acclimate to the context (conditioning chambers) before any behavioral testing.
	<ol>
		<li>Pre-expose the females to the conditioning chambers (located in sound-attenuating boxes) with the house lights on for 20-30 min per day for 3 days prior to any experimentation. Assign the box they are pre-exposed to as their chamber for the study. Thoroughly clean the boxes (walls and trays) between sessions and animals to remove odors since they can affect behavior and estrous cycle.</li>
	</ol>
	</li>
</ol>
4. Day 1: Habituation/Fear Conditioning
<ol>
	<li>Once the animals have been verified to be in the estrus phase of their estrous cycles, begin the habituation/conditioning phase of the 3-day experiment.
	<ol>
		<li>Take pre-tone baseline measures of freezing prior to the first tone conditioned stimulus(CS) presentations on each day of the experiment. Do this by acquiring percent freezing scores(calculated by dividing the seconds spent immobile by the duration of trial, multiplied by 100) during the stimulus-absent time interval at the beginning of the first CS-alone trial before the CS onset. 
		NOTE: These measures can be used to assess and compare baseline freezing levels with those during habituation, conditioning, extinction, and recall.</li>
		<li>To conduct extinction training during the metestrus phase, condition the rats in their last day of estrus. Swab and identify estrous phase (step 2) each day before starting the experimental protocol (conditioning/extinction/recall). As the estrus phase may last longer than one day, use cycle history to predict length of phases to determine when the animals are ready to begin training.</li>
	</ol>
	</li>
	<li>In the conditioning chambers, connect the foot-shock grid floor to an electric stimulation generator. Use a stimulation level of 0.5-0.6 mA as the unconditioned stimulus (US). Use a 4 kHz, 80 dB tone as the conditioned stimulus (CS). 
	NOTE: These parameters may vary across laboratories and may be modified for optimal conditioning.
	<ol>
		<li>If the freezing analysis program requires visual cues (as opposed to temporal), co-activate an LED light with the tone. Position the light on the outside of the operant chamber, within the video frame. At the start of each trial, activate the LED light and keep it illuminated until the end of the trial. 
		NOTE: This light is not visible by the animal and only serves as a marker for the initiation and end of the trial within the recorded video files (to be used to measure freezing behavior).</li>
	</ol>
	</li>
	<li>Place the animals into the conditioning chambers for 5 CS-alone trials of habituation. Immediately following the habituation session, perform 7 paired CS-US trials of fear conditioning. Ensure that the CS tone lasts for 30 sec and coterminates with the 0.5 sec US shock. Run trials in all sessions (habituation, conditioning, extinction, and recall) with a variable intertrial interval averaging 3 minutes.</li>
	<li>At the end of the conditioning session, return the animals to their home cages and to the animal facility until the next day.</li>
</ol>
5. Day 2: Estradiol Preparation
<ol>
	<li>Swab and identify the estrous phase for all animals that underwent Day 1 of this protocol. Prepare estradiol (also known as E2, 17beta-estradiol, or beta-estradiol) for a 15 &#956;g/kg dose subcutaneous injection using sesame oil or 0.9% saline as vehicle.</li>
	<li>To calculate the amount of estradiol to prepare for injections, use the body weights of all animals, which were measured and recorded when they were conditioned on Day 1 of this protocol.</li>
	<li>Make calculations to standardize the administered volume of estradiol (i.e. 0.2 ml) per rat. Since estradiol is difficult to dissolve due to its water insolubility and requires a large volume of solvent for the small dose, prepare a stock solution by mixing estradiol in sesame oil over heat until dissolved. The solution can then be passed through a 0.22 &#956;m filter to remove contaminants. For example, add a calculated volume of a 3,000 &#956;g estradiol/1 ml sesame oil stock solution to the total needed volume of sesame oil in order to achieve a 15 &#956;g/kg dose in a 0.2 ml injection volume per animal. 
	NOTE: Alternatively, use other forms of estrogen that are water-soluble and easier to dissolve.</li>
</ol>
6. Day 2: Estradiol Administration/Extinction Training
<ol>
	<li>Administer the estradiol subcutaneously. Lift the loose skin between the scapula by the neck in a gentle pinch and insert the syringe needle into the triangle that is formed by the skin folds.</li>
	<li>30 minutes following subcutaneous injections of estradiol, place the animals in the conditioning chambers again and begin extinction training. In this extinction session, perform 20 CS-alone trials, with each trial consisting of a 30 sec, 4 kHz, 80 dB tone, presented at a variable intertrial interval averaging 3 min. 
	NOTE: The effect of estradiol on recall occurs when estradiol is administered prior to extinction training or immediately after extinction. The effect is not observed 4 hr post-extinction, so it is recommended to inject close in time to the beginning or end of the training session15.</li>
</ol>
7. Day 3: Extinction Recall
<ol>
	<li>24 hr after the extinction training, swab the animals that completed Day 1 and 2 of this protocol.</li>
	<li>Place the animals into their assigned conditioning chambers (same as Day 1 and 2). Begin the extinction recall session by presenting 3 CS-alone trials (the three trials of extinction recall), which consists of only 3.80 dB CS-alone trials with intertrial intervals similar to the 20 trials administered for extinction training as described in step 6.2.</li>
	<li>Upon completion of the recall session, return the animals to their home cages.</li>
</ol>
8. Data Analysis
<ol>
	<li>Acquire behavioral data through video and analyze them using computer software. 
	NOTE: Freezing scores can also be manually counted by an experimenter that is blind to the drug treatment by timing freezing bouts during the CS presentation of each trial, expressed as percentage of the time spent freezing during the tone. Freezing is defined by complete lack of movement with the exception of breathing during the 30 sec CS-US trials. Calculate percent freezing by dividing the seconds spent freezing (immobile) by 30 seconds (duration of trial) multiplied by 100.</li>
</ol>

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The authors of this manuscript have no competing financial interests or other conflicts of interest to disclose.

Fear extinction has been studied extensively in male rats with the neural mechanisms mediating conditioned fear extinction identified and assessed under various manipulations. Relatively few studies have examined female rats or the role of gonadal hormones in fear extinction. To specifically study the effects of estrogen on fear extinction, naturally cycling female rats are subjected to a three-day behavioral paradigm. This procedure consists of habituation/conditioning, extinction, and extinction recall phases. Using th...

Inherent sex differences are observed across various cognitive behaviors and learning paradigms in both humans and rodents. For example, it has been reported that women generally have stronger verbal and detail-oriented abilities, whereas men have better spatial abilities1-3. These sex differences may be due in part to the influence of gonadal hormones. High estradiol levels improve performance on tasks in which women are better, but worsen performance on tasks that men typically do well4-6. Although this evidence is compelling, the limited ability to completely control experimental environments in human studies makes it difficult to determine if these effects on behavior can be attributed specifically to hormones. Animal studies, in contrast, allow for fully controlled circumstances.
Although the basic neural mechanisms of fear extinction have been identified and are well studied in males, it is unclear whether these systems are the same in females or how they change across the estrous cycle. Fear conditioning and extinction are widely used behavioral paradigms in rodents and humans to conduct studies related to anxiety disorders. Given that women have a higher risk for anxiety disorders as well as higher symptom duration and severity7-13, it is critical to include females in these studies. The underrepresentation of females in this research may be attributed to the challenges of estrous phase monitoring and accounting for the behavioral effects of sex hormones. Laboratories that do examine females in this regard encounter issues that often go unreported or are not described in the methods for their studies.
Emerging evidence in rodents suggests that sex differences in fear extinction are modulated by gonadal hormones14-18. In fear conditioning, animals are trained to fear a particular stimulus. After a number of unreinforced presentations of the stimulus, the animals learn not to fear the cue, a process called extinction. How well the animal learns and consolidates the memory of learning this task can be observed during an extinction recall test given some time after the extinction training. Less fear expression during extinction recall demonstrates good extinction memory consolidation. Recent findings from our laboratory suggest that estrogen can modulate the consolidation of fear extinction memory and improve extinction recall. Specifically, female rats that are extinguished in proestrus, the phase of the estrous cycle during which circulating estrogen levels peak, exhibit enhanced retention of extinction memory. In contrast, females that are extinguished in the low-estrogen metestrus phase display relatively poor extinction recall, which can be improved with exogenous estradiol administration prior to or immediately after extinction training15,16. The neural mechanisms of extinction memory consolidation (including the role of gonadal hormones) in females are not clear.
In laboratory animals, the role of hormones can be investigated using invasive surgical removal procedures such as castration and ovariectomy. Subsequent to recovery from surgery, gonadal hormones are often exogenously manipulated during the performance of a behavioral task19. This approach has provided critical information about sex hormones and is useful because it allows for well-controlled manipulations of gonadal hormones (in terms of timing and dose)20-23. This approach, however, does not assess the influence of the naturally occurring fluctuations that occur across the estrous cycle nor do they represent &#8220;normal&#8221; animals, thereby limiting the translational potential to human studies. It has been well-documented that female sex hormone levels peak and decline at specific phases of the estrous cycle, and estrogen receptor expression changes within the cycle, and after ovariectomy24. Thus, there is a need to conduct studies on females with intact gonads and refine experimental designs to reliably study the effects that high and low estrogen states may have on females throughout their lifespan.
This protocol focuses on the effect of estrogen on the neurobiological systems involved in fear extinction. It describes how to: 1) carefully monitor the estrous cycle, 2) prepare effective doses of estradiol for systemic administration, and 3) follow a behavioral paradigm that includes fear conditioning, extinction, and recall in naturally cycling female rats. This protocol can be modified with other pharmacological manipulations and cellular and molecular tools to aid studies to better understand sex differences that are observed in conditioned fear extinction behavior. Note that the procedures described below are those utilized within our laboratory, and there exist a number of variations of these procedures in the literature.

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			<td height="43" style="height:43px;width:120px;">Fear conditioning chamber</td>
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			<td height="43" style="height:43px;width:120px;">Selectfrost microscope slides</td>
			<td style="width:159px;">Fisher Scientific</td>
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			<td style="width:159px;">Becton Dickinson</td>
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			<td style="width:159px;">Exelint International, Co.</td>
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			<td style="width:199px;">26-gauge</td>
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protocol for studying extinction of conditioned fear in naturally cycling female rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

In this fear extinction recall protocol, percent freezing was measured as an indicator of fear. Animals that extinguished well and retained the memory of the extinction training exhibited low fear on the last day of the behavioral testing during extinction recall. Male and female rats do not significantly differ in conditioned fear expression during the conditioning, extinction, and recall phases (Figure 2). However, a sex difference becomes evident when the animals are analyzed separately as high- and l...

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Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

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13.2K Views.  Massachusetts General Hospital. Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Video: Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

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 ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

A Treatment Package without Escape Extinction to Address Food Selectivity

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including autism. Behavior analytic interventions for the treatment of feeding difficulties oftentimes include escape extinction as a primary component of treatment. The use of escape extinction, while effective, may be problematic as it is also associated with the emergence of challenging behavior (e.g., extinction burst). Such challenging behavior may be an acceptable side effect in treatment cases where feeding problems are severe and chronic (e.g., failure to thrive). However, in more acute cases (e.g., selective eating), the negative side effect may be unwarranted and undesired. More recent research on the behavioral treatment of food selectivity has begun to evaluate treatments for feeding difficulties that do not include escape extinction (e.g., demand fading, behavioral momentum), with some success. However, research to date reveals individual differences in responsiveness to such treatments and no clear preferable treatment has emerged. This manuscript describes a multi-component treatment package that includes shaping, sequential presentation and simultaneous presentation, for the treatment of food selectivity in four young children with developmental delays. This treatment package extends the literature on the behavioral treatment for food selectivity and offers a multi-component treatment protocol that may be clinically applicable across a range of treatment scenarios and settings.

Feeding difficulties are a common problem for children with developmental disorders, including autism, and behavioral interventions often include escape extinction. Recent research has begun to evaluate treatments that do not include escape extinction. This manuscript describes a multicomponent treatment package that does not use escape extinction to treat feeding difficulties.

Feeding difficulties and feeding disorders are a commonly occurring problem for young children, particularly children with developmental delays including ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Pavlovian Conditioned Approach Training in Rats

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, &#34;wanted&#39; stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the ...

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 ...

A Conditioned Place Preference Protocol for Measuring Incubation of Craving in Rats

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed incubation of drug craving. Here, we describe a morphine conditioned place preference (CPP) protocol for measuring the incubation of craving in rats. In this protocol, a CPP paradigm mainly employing somatosensory cues is used to establish a long-term reward memory of morphine. A three-chamber CPP box that differs in the texture of the chamber floor is constructed. First, the animals are tested for their baseline preference to the two side chambers for three consecutive days. Then, they are injected intraperitoneally with morphine/saline and put into their non-preferred/preferred chamber for 45 min. After 6 days of conditioning, their preference to the side chambers is tested for 15 min at different time points after the last conditioning session. With this paradigm, the reward memory of morphine could last for at least 18 days. To test whether the above-mentioned protocol can model increased craving, the number of entrances into the two side chambers are counted during the abstinence period. The results show that the entrances increased, suggesting that the CPP paradigm could mimic the incubation of craving. Future studies can employ this model to study neural mechanisms underlying long-term memory and incubation of craving.

Here, a morphine conditioned place preference (CPP) protocol is described to measure incubation of craving in rats.

A major cause of repeated relapses is a craving for the drug. Drug craving increases progressively during the abstinence period, a phenomenon termed ...