Financial support for this research was provided by Fundaci&#243;n Universitaria Konrad Lorenz - grant number 9IN15151. The authors would like to thank the Communications Department at Konrad Lorenz University for their help with recording and editing the video, in particular Natalia Rivera and Andr&#233;s Serrano (Producers). Also, Nicole Pfaller-Sadovsky and Lucia Medina for their comments on the manuscript, and Johanna Barrero, Dean at Corporacion Universitaria Iberoamericana, for institutional collaboration. The authors have no conflicts of interest.

The following protocol&#160;was&#160;approved by the Institutional Animal Care and Use Committee of Fundaci&#243;n Universitaria Konrad Lorenz (IACUC-KL). The universal declaration of animal rights issued by International League of Animal Rights, Geneva, Switzerland (1989), and ethical principles of experimentation with animals issued by ICLAS were respected.
1. Subject preparation
<ol>
	<li>Select male adult Wistar rats (n = 12). House them in groups of four per cage for three days of acclimatization, prior to the beginning of the training and testing protocol. Provide rats with free access to water throughout the experiment. Control the room temperature between 20 &#176;C to 25 &#176;C, under a 12 h light-dark cycle (lights on at 07:00 h). 
	NOTE: Rat strains had shown differential performance during fear conditioning. For instance, Schaap et al. (2013) reported that Wistar and Lewis strains showed longer durations of freezing behavior compared with Fawn Hooded and Brown Norway rats12. Thus, differences in pain and thermal threshold should be assessed to adjust the intensity and duration of shocks.</li>
	<li>Maintain rats at 85% of their free-feeding weights (normal weight between 350-400 g) by giving restricted food access at the same hour every day. Weigh rats every day at the same hour during the light cycle. Calculate the ad lib weight (100% weight) for three days before the start of extended fear-conditioning training. 
	NOTE: Animals used in the present experiment were tested on additional instrumental tests that are not reported in here. Food deprivation was required for those additional tests. This procedural variation is assumed as likely to expand the scope of the present procedure, as it suggests the potential for instrumental-fear combined tests. However, studies using only fear conditioning tests will not require food deprivation.</li>
	<li>Randomly assign subjects to one of the following groups: emotional testing 6 weeks after training (n = 6); emotional testing 48 h after training (n = 6).</li>
	<li>Perform training and tests at similar hours, during the light phase of dark-light cycle. Assign the animals to the same experimental chamber and maintain the same order of animals during training and testing. 
	NOTE: An additional control that could be implemented is counterbalancing the order of animals during training and testing phases. We recommend using this technique when multiples groups are assessed, or different tasks are applied across experiments, to reduce a possible effect of task-order on behavior.</li>
</ol>
2. Apparatus setting and shock calibration
<ol>
	<li>Clean all the internal surfaces of the experimental chamber and stainless-steel grid floor with 10% ethanol. Repeat before testing each animal.</li>
	<li>Connect the equipment to a computer using a USB cable and start the freezing detection system equipment: the CPU, the controlling cabinet, the infrared light, the aversive stimulator/scrambler, and the shock-intensity calibrator. 
	NOTE: Although this protocol was executed using commercially available instruments (Table of Materials), equipment and software of different brands can be used. The apparatus consists of an internal acrylic square chamber (29.53 cm x 23.5 cm x 20.96 cm, called the experimental chamber) embedded in a wooden box covered with plastic formic. The external doors allow the isolation of sound, noise or light (attenuating box doors). The camera is located laterally in the internal part of the external door. The internal acrylic box with floor metal grids (36 stainless-steel rods, each one of 3 mm diameter and spaced 8 mm, center to center) allows footshock delivery. In one of the internal-lateral walls, a speaker is located 6 cm from the floor to present an auditory cue.</li>
	<li>Connect the red and black clips of the shock intensity calibrator (i.e., positive and negative connectors) to two any different rods on the grid floor. Connect the USB cable to the corresponding port of the computer. Make sure to connect the red and black clips to bars separated by another bar. 
	NOTE: This section describes the shock intensity calibration process using a specific brand of equipment mentioned in the Table of Materials. However, the calibration process can be performed using different brands of equipment. It is recommended to calibrate the intensity of the shock in three sectors of the grid floor to verify that it is consistent. In addition, always remove fecal and urine residues from the grid floor to avoid interference during the delivery of the shock.</li>
	<li>Start the shock-intensity calibrator software (Table of Materials). Choose an intensity of 1.0 mA in the application by clicking on the range arrow. Then, change the Run/Stop switch to Run. 
	NOTE: We propose 1.0 mA based on our studies with rodent models in our lab and literature that reports a range from 0.75 mA&#160;to 1.5 mA as adequate for studies of fear conditioning33,34,35.</li>
	<li>Switch on the aversive stimulator or the equipment used to deliver the footshocks and look at the shock intensity displayed on the panel of the application. If needed, adjust the intensity to 1.0 mA using the knob on the aversive stimulator. 
	NOTE: Aversive stimulator should be set to &#8220;OUT&#8221; to appropriately test, calibrate, and run the experiment.</li>
</ol>
3. Freezing detection system calibration
<ol>
	<li>Close the experimental chamber and sound-attenuating box doors. Do not introduce the animal at this point, as it will be placed into the chamber after the freezing detection system calibration has been completed. Check that the light intensity inside the box is between 20 and 30 lux.</li>
	<li>Start the freezing detection system software and open the Experiment setup dialogue window. Enter the details of each subject (such as subject identification number, date and group) and load the file titled &#8220;Training protocol VFC.pro&#8221; (available at <a href="https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ">https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ</a>). 
	NOTE: Context and cue tests use a different program configuration; thus, make sure to use the correct file on each test. At this point the correct file corresponds to &#8220;Training protocol VFC.pro&#8221;. Remember that during test phases the file corresponding will be different to training session.</li>
	<li>Choose the corresponding camera(s) and check the Save Video option (if needed). Set the Motion Threshold to 100, and Min Freeze Duration to 30 frames. 
	NOTE: This Motion Threshold value is based on the size of the species used (based on number of pixels). Minimum Freeze Duration value is recommended by the manufacturer. These values are used to ensure proper recognition of the animal&#160;in the chamber.</li>
	<li>Verify that the live feed from the chosen camera(s) appears on the screen, together with the motion threshold graph and the timeline of the different stimuli that are presented during the training (e.g., sound and shock). 
	NOTE: Using a different brand, the equipment setup should offer the possibility to measure&#160;the movements of the animal to detect an &#8220;index&#8221; of motion that should allow comparisons on the amount of time the animal is moving or freezing. Another possibility is using a software that with only the video source (during or after the experiment) can detect the amount of time the animal is in motion or freezing, such as free software ImageFZ13, open-source toolbox in Matlab14, or a free classifier of animal behavior as JAABA15.</li>
	<li>Click the Calibrate option three times, while checking that the Motion Index remains below 100 (threshold). Then, set the equipment to lock by clicking on the corresponding button on the screen. 
	NOTE: This section describes a freezing detection system calibration process using a specific brand of equipment listed in the Table of Materials. As was mentioned before, the calibration process can be conducted using different brands of equipment (for a review of different options in equipment and software see Anagnostaras et al. 2010)16.</li>
</ol>
4. Extended fear conditioning training
<ol>
	<li>Transport the rats in their home cages, covered with a cloth, from the animal care facility to the behavioral training room in the laboratory. Avoid exposure to noise or stress-generating conditions during the transport of animals to the behavioral training room. If several animals are transported at the same time, only bring the animals to be tested and maintain other rats in a holding room to enhance experimental control. Gently handle the animals for 2 min before starting the training. 
	NOTE: In the protocol, the animals were handled each day for 2 minutes before behavioral training. Following handling, animals were introduced in the experimental chamber. We recommended to manipulate animals to make rats habituate to the researcher.</li>
	<li>Introduce the rat to the experimental chamber. Handle it gently by the base of its tail and place it on the middle of the chamber. Close the experimental chamber and sound-attenuating box doors.</li>
	<li>Start the session by clicking on the Record button. Let the rat acclimate to the chamber for 3 min. This 3 min period is the standard recommended by the equipment manufacturer and serves as a baseline and habituation time to the chamber.</li>
	<li>Deliver twenty-five tone-shock pairings (trials) with a 60 s Inter-Trial Interval (ITI), starting on minute 3 of the session. Present the tone (conditioned stimulus &#8211; CS; 90 dB SPL, 2000 Hz, 50-ms Rise Time) during the last 10 s of each ITI, and the shock (unconditioned stimulus &#8211; US) during the last 2 s of each ITI. 
	NOTE: Activation of the Record button is conditional on cameras being properly calibrated and locked.</li>
	<li>Remove the rat from the experimental chamber when the 28 min session is over. Return animals to the respective home cage. Transport the rats in their home cages covered with a cloth from the behavioral training room to the animal care facility.</li>
	<li>Repeat freezing detection system calibration (steps 3.1-3.5) and fear conditioning (steps 4.1 and 4.3) to train all the subjects. 
	NOTE: We strongly recommend recalibrating the detection system for each animal to ensure that the software maintains the same parameters when it processes information for freezing detection.</li>
	<li>Resting period: During this period, have the animals rest individually in their home cages. Monitor the weight of the animals twice per week during the 6 weeks of the incubation period. Gently manipulate each animal for two min while they are weighted.</li>
</ol>
5. Context test &#8211; single 10 min session
<ol>
	<li>After the training phase, expose the animals to the first memory test called context test. During this 10 min phase, expose the rats to the same context in which training took place but do not present cues or shocks. Transport the rats in their covered home cages (e.g., with a cloth) from the animal care facility to the behavioral training room. Keep in mind that animals were divided into groups, thus one group&#160;is tested 48 h after the training phase and the other group is tested 6 weeks after training (see Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60537/60537fig01.jpg" /> 
Figure 1: Timeline of the experiment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60537/60537fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Clean all the internal surfaces of the experimental chamber and stainless-steel grid floor with 10% ethanol. Repeat before testing each animal.</li>
	<li>Repeat freezing detection system calibration (steps 3.1 to 3.5). Open the Experiment setup dialogue window and load the file named &#8220;Context test protocol.pro&#8221;, which is available from <a href="https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ">https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ</a>. 
	NOTE: This file contains the setup for this experimental phase that consists of no shocks or tones.</li>
	<li>Introduce the animal to the experimental chamber. Handle it gently by the base of its tail and place it on the middle of the chamber. Close the experimental chamber and sound-attenuating box doors.</li>
	<li>Start the session by clicking on the Record button. During this single 10 min context-test session, no stimuli are presented (shock neither sound).</li>
	<li>Remove the subject from the experimental chamber when the 10 min session is over. Return the animals to their respective cages and transport the rats in their covered home cages from the behavioral training room to the animal care facility. Repeat steps 5.2-5.5 to test all the subjects.</li>
</ol>
6. Cue test &#8211;&#160;single 13 min session
<ol>
	<li>One day after the context test, have animals undergo the second test of memory called cue test. During this phase, the rats will be in a different context of training&#160;during 13 min.; cues (tones) are presented, but no shocks are delivered.&#160;Transport the rats in their home cages covered with a cover from the animal care facility to the behavioral training room. Test a group 72 h after the fear conditioning training, and test another group 6 weeks and one day after training (Figure 1). 
	NOTE: A different system of transportation (from the animal care facility to the experimental room) could be implemented to differentiate more the context and cue tests. Since the animals were transported to the training session and context test session in their home cages, a different transport cage, bedding and/or cover could be used during transportation to the cue test session.</li>
	<li>Clean all the internal surfaces of the experimental chamber and stainless-steel grid floor with 10% ethanol. Repeat before testing each animal.</li>
	<li>To change the visual context, insert the plastic surrounding wall to&#160;the experimental chamber.</li>
	<li>To change olfactory context, apply 1% acetic acid to a cotton-tipped swab, and place it in the metal tray below the grid floor17,18,19.</li>
	<li>Repeat the freezing detection system calibration (steps 3.1-3.5). Load the file named file &#8220;Cue test protocol.pro&#8221; file, which is available from <a href="https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ">https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ</a>. 
	NOTE: This file contains the setup for this experimental phase, which consists of delivery of the same tones presented during the training phase (CS), but in the absence of shocks (US).</li>
	<li>Introduce the animal to the experimental chamber. Handle it gently by the base of its tail and place it on the middle of the chamber. Close the experimental chamber and sound-attenuating box doors.</li>
	<li>Start the session by clicking on the Record button. During the single 13 min cue test session, the CS stimulus (tone) is presented 10 times, starting on minute 3 of the session. 
	NOTE: The first 3 min correspond to the baseline of this session, followed by 10 cue test trials (that is, 10 s each) delivered with 50 s ITIs in the absence of shocks. The delivery of tones is automatic, via using the previously loaded file.</li>
	<li>Remove the animal from the experimental chamber when the 13 min session is over. Return animals to the respective cage and transport them covered to the animal care facility. Repeat steps 6.2 through 6.5 to test all the subjects.</li>
</ol>
7. Data analysis
<ol>
	<li>Obtain the general activity index (i.e., motion index) that is derived from the video stream using the freezing detection system software. This software automatically transforms the motion index to provide the percentage of freezing time per session and the number of freezing episodes. Set the freezing threshold to the default Minimum Freeze Duration setting of the system (1 s = 30 frames).</li>
	<li>Use the additional custom-made program (file available from <a href="https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ">https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ</a>) to obtain:
	<ol>
		<li>Use the program to determine the percentage of freezing during the first three minutes of the training session (i.e., baseline freezing, since no shocks or tones were presented before or during that 3 min period) and during the first three minutes of the cue test session.</li>
		<li>Use the program to determine the percentage of freezing for each of eight 3 min bins of the training session.</li>
		<li>Use the program to determine the percentage of freezing during the cue presentations (i.e., freezing during the tones) and no-cue periods (intertrial intervals; ITIs), for both training and cue-test sessions.</li>
	</ol>
	</li>
	<li>To obtain these data, open the freezing detection system software.
	<ol>
		<li>Select File | Reports | Batch Component summary.</li>
		<li>Select the file with extension .CMP available from https://doi-org.bdigitaluss.remotexs.co/10.17605/OSF.IO/4NKFQ.</li>
		<li>Name the output file and change Motion Threshold to a 100. Then, click OK.</li>
		<li>Select the files to be analyzed (extension .RAW). These files are automatically generated from the freezing detection system software when the session is over and correspond to the raw data of each session. Initially, the files are saved in the desktop of the computer, but they can be stored in a custom folder (e.g., Documents-Fear conditioning) to facilitate their subsequent identification and opening when they need to be analyzed.</li>
		<li>Open the output files (extension .CSV). They can be edited in a spreadsheet software for further analysis. This file contains the results of freezing during the experimental session. 
		NOTE: To obtain the total percentage of freezing, divide the time that the subject spent immobile over the total session time. The number of freezing episodes can be calculated counting the number of freezing events through the session. In both cases, it is necessary to define a motion threshold based on a minimum freeze duration. This is the temporal criterion that defines whether a Freeze Episode is recorded. Automated systems of recording can use certain amount of frames per second (fps) as a measure of minimum freeze duration. For instance, with a sample rate of 30 fps, a minimum freeze duration of 15 frames will record as freezing an instance of immobility that last for 30 s.</li>
	</ol>
	</li>
	<li>Calculate the average duration of each freezing episode for each session (training and both tests, context and cue) by dividing the total freezing duration (in seconds) over the total number of freezing episodes.</li>
</ol>

The authors have nothing to disclose.

The present extended fear-conditioning protocol is an efficient and valid approach to assess emotional memory across short (48 h) and long-term periods (6 weeks). Thus, the protocol allows to study overtraining and fear incubation phenomena in rats. Among the different advantages of this protocol are the following. It offers two types of memory tests, namely context and cue, that allow to identify the differential effect of two delays (48 h and 6 weeks) across context and cue manipulations. Second, the task entails a sin...

Memory is a psychological process encompassing different phases: information acquisition, consolidation (allows for the stability of acquired information), and retrieval (evidence for the consolidation process)1. During the consolidation phase, the establishment of new synaptic connections and modification of pre-existing connections occur. This suggests the necessity for a period of time during which molecular and physiological events responsible for these changes occur1,2. These physiological or molecular changes vary whether the retrieved events are emotionally charged or not (i.e., emotional memory). For instance, research has shown that the lateral nucleus and basolateral amygdala complex are particularly relevant to emotional memory3,4,5.
Emotional memory phenomena have been primarily studied with fear conditioning paradigms5,6. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli7. Fear conditioning paradigms produce molecular, cellular, and structural changes in the amygdala. In addition, fear conditioning modifies the connectivity of the hippocampus during the consolidation and retrieval processes of emotional memory.
One of the most commonly used procedures for studying fear memories is classical (Pavlovian) conditioning in rats. This procedure typically uses footshock (US) as the aversive stimulus, which is delivered once or several times across one or several sessions. The conditioned response (CR) of rats exposed to this procedure is freezing (i.e., &#8220;generalized immobility caused by a generalized tonic response of the animals&#8217; skeletal musculature except those muscles used in breathing&#8221;7 ). This response could be assessed on two types of tests: context and cue tests. For the context test, the subject undergoes a given number of footshocks during the training session, and then is removed from the experimental chamber for a defined time. During the test, the subject is returned to the same context in which the training took place and different measures of freezing are collected in the absence of footshocks (e.g., duration, percentage or frequency of freezing episodes), and compared to baseline levels established during the training phase. For the second type of test, cue test, a stimulus (typically a tone) is paired with the footshocks during the training phase (i.e., conditional stimulus, CS). After training is completed, the animal is removed from the training context for a defined time and is subsequently placed in a modified context (e.g., a different experimental chamber that has different shapes of walls and different smell). The cue is then presented a given number of times, and freezing responses to the cue are measured and compared to baseline levels collected during training. The most common version of this paradigm uses 1 to 3 tone-shock pairings during a single training session, followed by context and cue tests conducted a number of hours or a few days later.
Other less frequently implemented fear conditioning procedures involve an extensive number of shock-cue pairings (i.e., trials), which have often been called overtraining procedures8. A growing interest in these tasks is related to their long-lasting and increased memory effects called fear incubation (i.e., conditioned fear responses increase over time in the absence of further exposure to aversive events or conditioned stimuli)9,10,11. An example of such overtraining procedures entails a training phase of 100 tone-shock pairings distributed across 10 sessions, followed by context and cue tests conducted 48 h and 30 days later11,12. To avoid extensive training spread across several days, Maren (1998) reported that overtraining could be established and optimized in a single session with 25 pairings8. The incubation effect is evidenced in significantly higher levels of conditioned fear in rats tested 31 days after training, as compared to rats tested 48 h after. Extended fear-conditioning tasks have been key for the understanding of behavioral and neurobiological aspects underlying fear incubation, including its relationship with other psychological phenomena (e.g., delayed-onset post-traumatic stress disorder)11,12,13.
Here, we describe an extended fear-conditioning protocol that induces overtraining and fear incubation in rats. Different to other paradigms that require several days of training11, the current protocol is focused on a single training session8. We used&#160;25 tone-shock pairings to produce higher conditioned freezing responses during context and cue tests conducted 6 weeks after training, as compared to tests conducted 48 h after.

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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.

Variations in percentage of freezing time during different stages of the training session were analyzed for all subjects (n = 12) using a dependent t test (Table 1). Animals were active and explored the experimental chamber during the first three minutes of the training session (first day of the protocol), time during which no tones or shocks were delivered (i.e., baseline-BL). As shown in Figure 2A, percentage of freezing time during th...

Watch this Scientific Journal Video about Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats at JoVE.com

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

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 ...

fear-incubation-using-an-extended-fear-conditioning-protocol-for-rats

Universidad San Sebastian

Research

JoVE Journal

Behavior

8.1K Views.  Universidad Pedagógica y Tecnológica de Colombia. 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.

Video: Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

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 ...

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.

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 ...

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

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

Neuroscience

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 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 ...

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. 
The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal ...

Studying Active Defensive Behaviors in Mice with Modified Fear Conditioning

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

Author Spotlight: Exploring Neural Correlates of Defensive Behaviors in Fear Learning and Extinction

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...

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 ...

Fear Conditioning

Fear Conditioning is a type of learning in which an association is established between a negative unpleasant event and a harmless stimulus. This leads to a fear of the harmless stimulus. This process is largely mediated by the amygdala, which is a brain region involved in emotions and stress reactions. Fear conditioning can be utilized in several ways to understand different aspects of learning and memory.
This video presents an overview of the principles behind fear conditioning, discusses the equipment and a generalized procedure used for this type of experiment. Finally, we'll review some real world applications of fear conditioning in behavioral neuroscience research today.

Fear Conditioning- Classical Conditioning: Principle and Procedure

Fear Conditioning is a type of learning in which an association is established between a negative unpleasant event and a harmless stimulus. This leads to ...