Name: Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice (Video) | JoVE Journal
Uploaded: 2014-03-01T15:45:00.000+00:00
Duration: 19 min 32 s
Description: Learn contextual and cued fear conditioning in mice using an automated video analysis system. This standardized protocol measures freezing behavior to assess fear learning and memory reliably.

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Some of the data shown here were obtained in the laboratory of Dr. Jacqueline N. Crawley in the U.S. National Institute of Mental Health and we would like to thank her for allowing us to show the data in the paper. We also thank Kazuo Nakanishi for his help in developing ImageFZ program for behavioral analysis. This research was supported by Grant-in-Aid for Scientific Research (B) (21300121), Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Science, Sports and Culture of Japan, grant from Neuroinformatics Japan Center (NIJC), and grants from CREST of Japan Science and Technology Agency (JST).

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We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

The contextual and cued fear conditioning test is one of the most widely used paradigms to assess learning and memory. This test is a form of Pavlovian conditioning in which an association is made between a context and/or a conditioned stimulus (auditory cue) and an aversive stimulus (electric footshock). After even a single pairing of the context/auditory cue and footshock, mice exhibit long-lasting freezing when faced with either the context or the cue. In this test, freezing behavior is used as an index of fear memory. Pharmacological and lesion studies have revealed that memory formation, consolidation, and retrieval are regulated by several brain regions, such as the amygdala, hippocampus, and prefrontal cortex3,33-35. In addition, molecular genetics studies have demonstrated the role of specific genes and molecules involved in learning and memory in these brain regions using genetically engineered mice36. Therefore, this test is simple and useful for exploring the neurobiological basis underlying fear learning and memory. In this movie article, we introduced our protocol to provide experimenters with detailed information to understand and easily perform the test.
Freezing behavior was quantified through direct observation by human experimenters. A well-trained experimenter is expected to produce reliable, stable results across observations. However, this method involves potential problems, such as differences in the observational method, observer biases, and simple quantification mistakes, making it difficult to directly compare the results from independent experimenters and different laboratories. An automated photobeam-based computer measurement system has also been used26,30-32. However, this system also presents potential problems in measuring freezing behaviors. Because of the sensor arrangement, this system might be unable to detect small head movements that would typically be scored as &#8216;active&#8217; through human observation. In addition, trembling during freezing might be considered as nonfreezing because when an animal freezes, intermittent interruptions in the photobeam are observed as a consequence of trembling. As an alternative method, automated image- and video-analyzing systems have been developed17-20,37,38. Anagnostaras et al.37&#160;described a few systems with image analysis software programs that have good validity and score freezing well17,20,37-38. However, most of these systems and analysis programs have to be obtained from commercial suppliers and are typically costly. We developed the ImageFZ software program for the analysis of freezing behavior, and this program is distributed as a free software program. ImageFZ detects the mouse as a body of pixels (a particle) and discriminates subtle mouse movement as &#8216;freezing&#8217; or &#8216;non-freezing&#8217; depending on the amount of area of nonoverlapping regions between particles of each pair of consecutive images. As shown in the representative results, measurements using the ImageFZ program are consistent with or more accurate than obtained using other methods. Thus, the ImageFZ program automatically measures the behavior that human observers judge as freezing using defined criteria. In addition, the ImageFZ program calculates the distance traveled (cm) before, during, and after footshock exposure, facilitating an assessment of the shock sensitivity and analysis of the freezing behavior.
Methodological differences exist between laboratories. These differences may result in difficulty in comparing data among laboratories and in replicating results in different laboratories. To obtain more stable and comparable data, it is necessary to standardize the test protocol as much as possible. The analysis system with ImageFZ leads to the automation of test procedures, which can contribute to the standardization of protocols used across laboratories.
Several behavioral responses must be considered when analyzing freezing behavior. First, when animals face a fearful situation, they may flee instead of freezing39. Fleeing is one of the fear responses, and its occurrence will lead to underestimating fear memory. Second, freezing may depend on a general activity level, and the activity level in experimental and control mice needs to be examined. For example, although mice lacking the M1 muscarinic acetylcholine receptor showed reduced levels of freezing compared to wild-type mice, various behavioral tests indicated that the results may be attributed to their hyperactivity phenotype instead of their memory impairment18. ImageFZ calculates the distance (cm) traveled by the subjects. The data are available to examine whether or not differences exist in the general activity levels between subjects. If there is a group difference in the distance traveled, one possible approach to the problem is to consider the distance traveled during the first 2 min of training as the baseline activity and to use a suppression ratio (suppression ratio = (activity during testing)/(activity during baseline + activity during testing)) as a secondary index of fear17,40. Finally, a difference in pain sensitivity, inducing the changes in reactivity to an electric footshock, if any, may result in variations in freezing behavior. ImageFZ also calculates the distance traveled (cm) in detail from 2 sec before an exposure of a 2&#160;sec footshock to 2 sec after its exposure (for 6 sec), which can be used as an index of footshock sensitivity.
Video-analyzing systems have been developed to measure the freezing behavior of albino, black, agouti, and dilute brown mice. ImageFZ uses a black floor tray and black grids to examine white mice (see Figure 1B). The black grids are made of specially processed metals with coated black paint and have an electrical conductivity similar to that of the noncoated metal grids, which are typically used for black mice. ImageFZ also analyzes the freezing behavior in rats and other rodents through adjustments of the program parameters. In the current version of ImageFZ, the behavior of the subject is recorded using a video camera from the top wall to analyze freezing. ImageFZ could also be used in a set-up where the images are captured from the side of the chamber. In addition, the ImageFZ controls a maximum of 4 apparatuses. This feature allows the researcher to simultaneously examine 4 mice, saving time and reducing the potential influences from differences in the execution time of each subject and the testing order on behavior. Thus, ImageFZ simplifies the testing procedure and analysis of freezing behavior, and this program facilitates testing with less labor and without any training for the behavioral experiments.
In the Miyakawa lab, we have assessed more than 110 strains of genetically engineered mice and wild-type control mice in the contextual and cued fear conditioning test using the video analyzing system to elucidate the effects of a given gene on learning and memory41-42. We have obtained a large set of raw data for more than 5,000 mice. The raw data that was used for published research articles4-16 are included in the &#39;Mouse Phenotype Database&#39; as a public database (URL: http://www.mouse-phenotype.org/). The present movie article provides detailed information regarding the details of our experimental procedure and promotes the understanding of the testing situation.

The contextual and cued fear conditioning test is the behavioral paradigm used to assess associative fear learning and memory in rodents1-3. This test has been widely used to understand the neurobiological mechanisms of fear learning and memory in transgenic and knockout mice1,4-16. Freezing behavior, which is defined as complete immobility with the exception of breathing, is a common response to fearful situations. In this behavioral paradigm, after animals are exposed to a pairing of an auditory cue with an electric footshock, they respond to the fear-producing stimulus by displaying freezing behavior, which is measured as an index of associative fear learning and memory. This test requires less elaborate equipment, less physical exertion by the investigator, and much less training time for mice than other learning and memory tasks; it generally requires approximately 5-10 min/day per mouse for 2 days. Although the testing procedure is simple and requires little time to perform, the investigator must carefully observe and measure mouse behavior; therefore, several automated measurement systems have been developed to conduct the behavioral analysis17-20. Our video-analyzing system, which we developed with the ImageFZ software program, allows us to easily analyze freezing behavior and produce highly reliable results. This article provides detailed information on our testing procedure and describes how to use the ImageFZ software program.

<table><tbody><tr><td>ImageFZ program</td><td>Developed by Tsuyoshi Miyakawa</td><td></td><td>This program is available through O&#039;Hara &amp;amp; Co., Tokyo, Japan and for free download at http://www.mouse-phenotype.org/. This software runs on 32-bit Windows XP/Vista/7.&amp;nbsp;</td></tr><tr><td>Conditioning chamber</td><td>O&amp;rsquo;Hara &amp;amp; Co., Japan</td><td>CL-3002L</td><td>For mouse.</td></tr><tr><td>Cued test chamber</td><td>O&amp;rsquo;Hara &amp;amp; Co., Japan</td><td>CLT-3002L</td><td>For mouse.</td></tr><tr><td>Interface</td><td>O&amp;rsquo;Hara &amp;amp; Co., Japan</td><td>CL-1040</td><td>The interface includes a white noise/tone generator, which can be controlled by ImageFZ program.</td></tr><tr><td>Scrambled shock generator</td><td>O&amp;rsquo;Hara &amp;amp; Co., Japan</td><td>SGA-2040</td><td>The shock generator can be controlled by ImageFZ program.</td></tr><tr><td>Shock grid tester (ammeter)</td><td>O&amp;rsquo;Hara &amp;amp; Co., Japan</td><td>SG-T</td><td></td></tr><tr><td>USB video capture device</td><td>XLR8</td><td>USB2IVOSX</td><td></td></tr><tr><td>Quad image splitter</td><td>Wireless Tsukamoto Co., Ltd., Japan</td><td>400AS</td><td></td></tr><tr><td>Soundproof room</td><td>O&amp;rsquo;Hara &amp;amp; Co., Japan</td><td>CL-4210</td><td></td></tr><tr><td>Freeze Monitor</td><td>San Diego Instruments, Inc., CA, USA</td><td></td><td>16 x 16 photbeam array&amp;nbsp; ( 2.5 cm spacing)</td></tr></tbody></table>

contextual and cued fear conditioning test using a video analyzing system in mice

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.

In the fear conditioning test, human experimenters used to quantify the freezing behavior through labor-intensive direct observation26-29, but recently photobeam-based computer measurement (e.g. the 'Freeze Monitor' system) and image-analyzing systems have been used to automatically measure the freezing behavior26,30-32. ImageFZ is an automated image-analyzing system, which produces results comparable to those obtained through human observation, as described below. Here, we compared the outcomes of human observation with those of ImageFZ analysis under varying parameters: 'Rate (frame/sec)' and 'Freezing criterion (pixels).' In this experiment, five male C57BL/6J mice (mean body weight ± SD (g), 31.4±3.55; mean body size ± SD (pixels), 351.6±62.2) were used at 15-27 weeks of age. The human observation was made using an event-recording program (a Macintosh OS9 software program); a key-pressing event that continued for 2 sec or more when a mouse displayed a bout of no movement was considered 'freezing'. The percentage of freezing was calculated every 60 sec in each test and used for correlation analyses. The percent of freezing scored by the 2 observers (interobserver reliability, for conditioning, r=0.879; for context test, r=0.957; for cued test, r=0.866, for all cases, r=0.888) was averaged to generate a human score. Correlations between the freezing percentages measured through ImageFZ with each frame rate (i.e. 1, 2, and 4 fps) and those obtained through human observations were examined. As illustrated in Figure 4, the freezing percentages calculated through ImageFZ (1, 2, and 4 fps) were highly correlated with the average value obtained from the measurements of the 2 observers. Notably, capturing images at a higher frame rate does not always produce the best correlation. Image analysis at 1 fps generated results similar to those obtained from human observers in each test. Correlations between the freezing percentages measured through human observations and using ImageFZ under each condition of the 'Freezing criterion (pixels)' (i.e. 20, 30, and 40 pixels) were examined. The freezing percentages calculated using ImageFZ at the 'Freezing criterion (pixels)' of 20, 30, and 40 pixels were, in all cases, highly correlated with those obtained through human observations (Figure 5). As shown in Figure 5D, when the freezing criterion is set to a low value, the subtle movement of a mouse, considered to be 'freezing' by human observers, would be considered 'non-freezing' using ImageFZ. Conversely, if the criterion is set to a high value, the movement of a mouse, scored as 'non-freezing' by human observers, would be considered 'freezing' using ImageFZ (Figures 5C, 5F, and 5I). Thus, to obtain the most reliable results, each parameter of the ImageFZ program should be calibrated using the data scored through human observations in each testing environment.
In addition, we compared the results generated by a human observer, using a photobeam-based computer measurement system (the Freeze Monitor system), to those obtained using ImageFZ (see Figure 6). The human observer was blinded to the treatment group and the results of ImageFZ scoring. For the parameter settings of the Freeze Monitor system, we used 3 measures of the percentage of freezing from a previously validated system30. Briefly, the number of 10-sec intervals in which the animals required more than 1 or 2 sec to cross the first new beam of the interval (1sec 10sec and 2sec 10sec, respectively) and the latency between the beginning of each 5 sec interval and the third new beam interruption within this interval (Latency3) were measured. The percentages of the intervals during which the mouse was freezing or the percentage of the total amount of time required to break the third photobeam were calculated.
The percentages of freezing measured in each system are illustrated in Figure 6. The groups were compared using two-way repeated measures ANOVA followed by t-tests (see Table 1). The freezing percentages measured using ImageFZ (Figure 6B) were more similar to those scored through human observation (Figure 6A) than the data obtained using a photobeam-based system (Figures 6C-E). The freezing percentages measured using the ImageFZ program in each test were highly correlated with those scored through human observation (conditioning, r=0.947; context test, r=0.970; cued test, r=0.934), whereas the correlations between the freezing percentages measured using the photobeam-based computer measurement system (1sec 10sec, 2sec 10sec, or Latency3) and the human observer were lower (conditioning, r=0.503, 0.593, and 0.761; context test, r=0.772, 0.819, and 0.912) compared to the correlations between the freezing percentages measured using ImageFZ and human observation (Figures 7A and 7B). In addition, Figure 7 reveals that the differences between the freezing percentages obtained through human observation and using ImageFZ in each mouse were the smallest differences. These results indicated that the freezing percentages measured using ImageFZ were similar to those obtained through human observation and that ImageFZ is highly accurate when measuring the amount of freezing.
<img alt="Animal behavior experiment setups showing chambers, grids, shock generator, and soundproof room." fo:content-width="5in" fo:src="/files/ftp_upload/50871/50871fig1highres.jpg" src="/files/ftp_upload/50871/50871fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Apparatuses for the contextual and cued fear conditioning test. (A) An acrylic square chamber for the conditioning and context test, (B) metal grids on a white plastic floor for black, agouti, or dilute brown mice (top) and electrifiable black metal grids on a black plastic floor for white mice (bottom); enlarged images of the grids are shown in the right panel, (C) a white noise/tone generator and a shock generator, (D) a soundproof room, and (E) an acrylic triangular chamber with a flat floor for the cued test. <a href="https://www.jove.com/files/ftp_upload/50871/50871fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Contextual and cued fear conditioning test diagram; CS/US presentation, experiment setup, timeline." fo:content-width="5in" fo:src="/files/ftp_upload/50871/50871fig2highres.jpg" src="/files/ftp_upload/50871/50871fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Schematic representation of the protocol. (A) Overview of the contextual and cued fear conditioning test, (B) conditioning, (C) context test, and (D) cued test. <a href="https://www.jove.com/files/ftp_upload/50871/50871fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Motion analysis process using binary thresholding and XOR for freezing behavior; experiment diagram." fo:content-width="5in" fo:src="/files/ftp_upload/50871/50871fig3highres.jpg" src="/files/ftp_upload/50871/50871fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Image analysis by the ImageFZ software program. For each pair of successive images, the amount of area (pixels) through which the mouse moved is calculated by ImageFZ. When this area is below a certain threshold (e.g. 30 pixels), the behavior is judged to be 'freezing'. When the amount of area equals or exceeds the threshold, the behavior is considered to be 'non-freezing'. <a href="https://www.jove.com/files/ftp_upload/50871/50871fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Static equilibrium; scatter plots; human observation vs ImageFZ rates; correlation analysis results." fo:content-width="6in" src="/files/ftp_upload/50871/50871fig4highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. Comparisons of the freezing percentages calculated from images at different frame rates using ImageFZ with those measured through human observation. The fear conditioning tests were conducted using male C57BL/6J mice (n=5). During the tests, two observers scored the freezing behavior. Simultaneously, live images were captured at 4 fps using the ImageFZ program. The files captured at 4 fps were downsized after extracting the frames to correspond to images captured at 1 fps or 2 fps . The parameter values of 'Rate (frame/sec)' were set to 1, 2, or 4 fps , and freezing percentages in each 60-sec bin were calculated from image files using ImageFZ offline analysis. Each dot represents a freezing percentage of each 60-sec bin. Pearson's correlation coefficients between the data obtained from human observation and ImageFZ analysis were calculated. <a href="https://www.jove.com/files/ftp_upload/50871/50871fig4highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Correlation scatter plots for ImageFZ vs. human observation under conditioning, context, cued tests." fo:content-width="6in" src="/files/ftp_upload/50871/50871fig5highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
Figure 5. The freezing percentages calculated from the images at different freezing criterion values using ImageFZ and those measured through human observations were compared. The fear conditioning tests were conducted using male C57BL/6J mice (n=5). During the tests, two observers recorded the freezing behavior, and the live images were captured using the ImageFZ program. The freezing percentages in each 60 sec bin were calculated from the images (1 frame/sec) through ImageFZ offline analysis, setting the parameter values of 'Freezing criterion (pixels)' to 20, 30, or 40 pixels. Each dot represents a freezing percentage of each 60-sec bin. Pearson's correlation coefficients between the data obtained from human observation and the ImageFZ analysis were calculated in each test. <a href="https://www.jove.com/files/ftp_upload/50871/50871fig5highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Graphs comparing freezing behavior in conditioned vs unconditioned subjects over time." fo:content-width="5in" fo:src="/files/ftp_upload/50871/50871fig6highres.jpg" src="/files/ftp_upload/50871/50871fig6.jpg" data-altupdatedat="1787630408000" data-alt="Figure 6"> 
Figure 6. The percentages of freezing were measured using automated systems and human observation in unconditioned and conditioned groups of male C57BL/6J mice (n=5, each group). (A) Human observation, (B) ImageFZ, (C) Freeze Monitor system 1 (1sec 10sec), (D) Freeze Monitor system 2 (2sec 10sec), and (E) Freeze Monitor system 3 (Latency3). Group comparisons were performed using two-way repeated measures ANOVA followed by t-tests (unconditioned group vs. conditioned group, *, P&lt;0.05; †, p&lt;0.01). The data obtained using ImageFZ were similar to those scored through human observation. <a href="https://www.jove.com/files/ftp_upload/50871/50871fig6highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Correlations and histograms comparing ImageFZ, Freeze Monitor, and human observations in freezing analysis." fo:content-width="4.3in" src="/files/ftp_upload/50871/50871fig7highres.jpg" width="420" data-altupdatedat="1787630408000" data-alt="Figure 7"> 
Figure 7. Correlation and frequency distribution of the differences between the freezing percentages, measured using automated systems and human observation. (A-B) Scatter plots and Pearson's correlation coefficients between freezing percentages scored through automated systems and human observation are shown. The freezing percentages, calculated using ImageFZ, were highly correlated with those obtained through human observation. (C-F) Occurrences of less than a 10% difference between the freezing percentages obtained from automated systems vs. human observation were highest when the data analyzed using ImageFZ were compared with those analyzed through human observation. <a href="https://www.jove.com/files/ftp_upload/50871/50871fig7highres.jpg" target="_blank">Click here to view larger image.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>ANOVAs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Condition</td>
			<td>Time</td>
			<td>Condition x Time</td>
		</tr>
		<tr>
			<td>Day 1 (conditioning)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human</td>
			<td>F(1,8)=28.53, p=0.0007</td>
			<td>F(7,56)=20.79, p&lt;0.0001</td>
			<td>F(7,56)=16.58, p&lt;0.0001</td>
		</tr>
		<tr>
			<td>ImageFZ</td>
			<td>F(1,8)=13.97, p=0.0057</td>
			<td>F(7,56)=21.40, p&lt;0.0001</td>
			<td>F(7,56)=11.69, p&lt;0.0001</td>
		</tr>
		<tr>
			<td>Freeze Monitor (1sec10sec)</td>
			<td>F(1,8)=5.16, p=0.0528</td>
			<td>F(7,56)=2.39, p=0.0329</td>
			<td>F(7,56)=0.72, p=0.6572</td>
		</tr>
		<tr>
			<td>Freeze Monitor (2sec10sec)</td>
			<td>F(1,8)=4.07, p=0.0782</td>
			<td>F(7,56)=3.44, p=0.0039</td>
			<td>F(7,56)=1.52, p=0.1803</td>
		</tr>
		<tr>
			<td>Freeze Monitor (Latency3)</td>
			<td>F(1,8)=4.44, p=0.0682</td>
			<td>F(7,56)=9.94, p&lt;0.0001</td>
			<td>F(7,56)=4.33, p=0.0007</td>
		</tr>
		<tr>
			<td>Day 2 (context)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human</td>
			<td>F(1,8)=42.94, p=0.0002</td>
			<td>F(4,32)=1.91, p=0.1336</td>
			<td>F(4,32)=1.48, p=0.2302</td>
		</tr>
		<tr>
			<td>ImageFZ</td>
			<td>F(1,8)=49.61, p=0.0001</td>
			<td>F(4,32)=2.06, p=0.1087</td>
			<td>F(4,32)=0.83, p=0.5174</td>
		</tr>
		<tr>
			<td>Freeze Monitor (1sec10sec)</td>
			<td>F(1,8)=20.28, p=0.002</td>
			<td>F(4,32)=1.63, p=0.1918</td>
			<td>F(4,32)=0.55, p=0.6997</td>
		</tr>
		<tr>
			<td>Freeze Monitor (2sec10sec)</td>
			<td>F(1,8)=40.20, p=0.0002</td>
			<td>F(4,32)=2.66, p=0.0504</td>
			<td>F(4,32)=1.20, p=0.3306</td>
		</tr>
		<tr>
			<td>Freeze Monitor (Latency3)</td>
			<td>F(1,8)=35.30, p=0.0003</td>
			<td>F(4,32)=2.49, p=0.0626</td>
			<td>F(4,32)=1.09, p=0.3793</td>
		</tr>
	</tbody>
</table>
Table 1. Comparisons of statistics.

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 ...

The contextual and acute to fear conditioning test is a behavioral test that is widely used to assess the ability of Lawrence to learn. Remember associations between an aggressive experience and environmental cues. However, in this test there are some methodological differences between the workplace, which may result in the difficulty in comparing data and in duplicating results in.To obtain more stable and tive data, it's important to standardize the test protocol as much as possible. In this we demonstrate protocol of the field test. Fear Functioning test is a simple, sensitive and reliable test for assessing your memory.This test is often used to understand neuro such and molecular mechanism and underly learning and memory, and also to evaluate cognitive function of potential mouse models of psychiatric disorders such as post stress disorder and schizophrenia. In the test facing behavior, which is measured as an index of learning and memory is scored by ization of human observers, was some automated systems to analyze the using behavior automatically. We develop an image analysis system using image FG and image A based software program.We are distributing such software program and it can be free downloaded at this address. In this we introduce our testing protocol and the automated analyzing system. First, we show how to safety the app operator as image of the software program.Second, we demonstrate the detailed protocols of the test, including conditioning context test, and Q test for device using the system. Finally, we present a present results which indicates that the N image FD system can produce highly reliable results. Now let us begin.Begin in the fear conditioning test fast. A mouse is placed in a chamber with grade floor. The OID is presented as an auditor.Auditor queue for 30 seconds. Thereafter, an electric code shock is provided. This pairing one auditor queue and the foot shock is given three times to enhance the association between the auditor queue and the electric code shock.During the test, the freezing behavior is measured as an index of associated learning and unfair memory. Second, after 24 hours, the mouse is placed into the same conditioning chamber and it's scored for fleaing behavior. To measure contextually control fear, the mouse is then transferred into differently shaped G without any auditory queue and thereafter, the auditory queue is presented.In this test, the freezing behavior is used as an index of acute fear memory. During the test, mouse behavior is recorded through a camera connected to a computer. The behavior is judged as freezing or non freezing.By the image software program, the program can calculate the percentage of freezing time and distance traffic. Here on example, image analysis is demonstrated during the test. Live images are captured at one length per second.Image of the creates a binary image from each live image for each pair. With successive flames, the amount of area through which the mouse moved is calculated by the image of the program. When the area is below a certain threshold, the behavior is judged as freezing, which is generally defined as a complete absence of any movement except for respiration and hard to beat.Freezing behavior that last less than the defined time threshold is not judged to be freezing. The threshold value must be set beforehand so that it yields the best fit between freezing times as measured by image Z and human observations. When the amount of area is equal to the threshold or X three Z, the behavior is considered to be non freezing.Here we demonstrate our test protocol in mice using this video analyzing system. In this protocol, the operators is constructive on a grid square chamber with metal grids and uncovered with a transparent lead image. FD can distinguish dark subject from a white background in each captured video image.But this process enables us to analyze the subject freezing behavior that the chamber needs to be placed on a white floor to analyze the behavior of black, a good or direct black mice. The freezing behavior of aluminum mice can also be analyzed by image D using black metal grids Under a black rule, light emitting diode lights are attached to the sail above the apparatus. The grid rule is illuminated at 100 roofs by the LED lights.A speaker connected with the white noise tone generator is mounted on sail above the lid. A 55 white noise is presented as an auditory queue. The grids are wired to a shock generator to deliver on electric food shock.The shock wind test is set at 0.3 million pair before the test is initiated. The current intensity on the grid should be checked by a metal because the shock test may be decreased by a buildup of fe and urine ro the mice, the test chamber is placed in soundproof loom to minimize external noise. During the test, the appellate is located in a different sound from the one where the coning and context tests are performed.It is composed of a chamber that has different properties from the coning chamber. Providing a normal context, it is necessary to change the sensory codes as much as possible so that the mouse will perceive Nobel context as unrelated to the conting chamber. In this protocol, the apparatus is an acri triangular chamber with a flat acri rule and the transparent lead.A speaker is mounted on sail above the lead to present on or three queue. This queue is the same cue that was provided to the mice at hawing. The illumination level of the floor is set to study looks.Each chamber is a cube with a sail mounted charge cab, device camera to monitor the mouse behavior. Before each test begins, the acrylic walls and floors are wiped with a tongue so in super high gross water and the grids are cleaned with 70%ethanol. To prevent a bias based on olfactory cut, the grids are wiped with Ethan instead of super hype gross water to inju that the grids don't deduce their electrical conductivity due to last two to four mice, A house package in a temperature controlled holding room to reduce the possible increases of cage transportation on behavior and to adapt the mice to the experimental environment.The cage containing the mice transferred into a soundproof waiting room at least 30 minutes before each test begins. All of the experiments should be performed during the same time period to minimize the behavior variations that can be produced by testing at different times. Mice from each group should be tested in a counter balance to order to reduce potential effects of the experimental time.Under the testing order of subject on behavioral performance, image D can control a maximum of four apparatuses testing from mice simultaneously while the researcher to save time and digital to deduction in the potential effects of the experimental procedure on mouse behavior. Before the continuing session begins, run the A FD application software. Select plug menu FD concerning and FD online and set the parameter values set by step as follows.Step one, project Id specify of folder where you want to store your date of files. Step two, session name type. Any words in the session box is the experimental date and the ference text to file in which the start time and the duration of the white noise and the food shock are written in the reference box.The deference text to file must be prepared in the deference folder in the image, the folder beforehand. Step three parameter settings. Enter the parameter values such as duration, time, and freezing criteria as shown in the movie.Step four, subject id. Enter the subject identification. Step five, camera settings contrast the brightness and contrast of the captured image.Step six, threshold settings. First to measure distance travel from the consecutive images, adjust threshold main value of the program, which is set to segment the images into black particle and white background. The distance travel is calculated from between each set with XY coordinates for center of gravity with the particle in the consecutive images.Second to major for behavior from the consecutive images, just assuming x extra value of the program which is set to segment the images into black particle under background and then calculate the amount of area of non-overlapping regions between particles of which pair of consecutive image intake window the non-overlapping region is shown. Change express means to value on the amount of value of the region should be below the ING cry value when mouse show ing behavior. These value should be adjusted so that the data by image fatigue matches that squad by human itself.Step seven, set to catch field specified the field, which chamber that you want to capture after clicking with the rectangle button in the toolbox to rectangular around the floor of it. Chamber on the live image window. Next selected chamber novel and click the set button to finally click the complete button.After the parameter settings have been defined, preparatory test should be given using practice mice before the first test of the date to determine whether the image analyzing system and what noise shock generators work with that problems move the comb cage contains the practice mice to the sound testing gloom from the adjacent to waiting gloom and place each mice into the conditioning chamber immediately after placing the mice into the chamber, click the start bottom of the image of Z.The mice are allowed to fully explore the chamber for 120 seconds.Thereafter. A white noise as an auditory queue is presented for 30 seconds and during the last two seconds of the sound, a food shock is given to the mice. This procedure is repeated three times in session to strengthen the association between the queue and the food shock.The masks are left with the chamber for 90 seconds after the last pairing. To further establish the association between the context of the chamber and the passive experience. After 480 seconds has elop, the mouse are returned to the home cage and the cage is returned to the shelf of the holding room.After each test, clean the chambers and grid carefully. Then click the next analysis button and repeat the session in the test mice. Approximately 24 hours after the concerning move the cage continu to mice to a soundproof waiting loom room.The holding loom at least 30 minutes before each test begins. Long image of the and set the parameter values of the software in the same manner as in the conditioning. However, set the duration time up to 300 seconds and select 35 text to file for the contextual test.35 text to file is blank. Place the mouse into the conditioning chamber and click the start bottom. The mice are allowed to fully explore the chamber for 300 seconds without any stimulus presentations.After 300 seconds, hassed li the mice into their home cage and clean the chambers. Then click the next analysis bottom and repeat the procedure in the test mice long image of D and set the parameter values in the same manner as in the continuing however ference text to file for this test and set duration time up to 360 seconds. This is a reference text to file for this test.Place each mice into the triangular chamber and click the start button of image Z.The mice are all to explore the chamber in the first three minutes. Neither white noise nor foot shock are presented. This chamber has different properties that provide a new context and related to the continuing chamber.In this chamber must typically move around a lot and don't show ing behavior. At the end of the first three minutes, the white noise that is presented at Concho is provided to the mice for three minutes. During the presentation of the auditor, auditor queue mice or exhibit written behavior after 360 seconds has erupt, returned the mice into their home cage and return the cage to the shelf of the holding room.After the test, clean the chambers carefully. Then click the next analysis bottom and repeat the procedure using the test mice. This video shows that the passage of freezing analyzed by the image of the system appears to be more closely related to thought measured by a human observer.The observation was confirmed by the details which showed that the percentage of leasing measured by image physi in each test were highly correlated or thought score by the human observer. In addition, this figure demonstrates that the differences between the freezing percentages measured by human observations and the image of the system in it image was the smallest. These results indicated that the percentage were freezing measured by the image of the system was similar to those obtained by human observation and that accuracy measuring the amount of reason by image Z is high.Okay.How did you like it? As you see the test, standard behavioral analysis can be performed easily. There are some tips and important points for using the image of the system, so let me ask ika about it.Okay.First, how did you feel about the testing procedure of the image of the system? I think our testing procedure is very simple and image of the system is a useful tool to measure reason, behavior accurately and quickly. Anyone can easily use a software program with a prior training As a result, this system can be used to assess their memory in many months in a short time, which will save us time and later.I See. Then is there anything we should be careful when we conduct the test? Yes, because The operator stating it is important to get sharpest image to measure with behavior precisely, and so you should adjust the camera length and the writing things belo.Also in the program study, you need to set the sexual values and reason criteria carefully to judge whether or not the behavior can be ified as reason. It is important that result was immediate, the program as with thought of them by human observation As shown in the the present result.Okay. As you can see, this may be article, provide detailed information regarding the test procedure and the use of the software program.We hope that this may be article. Promote your understanding of the test and contribute to the standardization of the protocol.

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Research

JoVE Journal

Behavior

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.

Trace Fear Conditioning in Mice

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.

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

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.

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

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.

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

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.

Observational Fear as a Model of Affective Empathy in Mice

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.

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

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

Quantifying Social Motivation in Mice Using Operant Conditioning

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm.

Reducing State Anxiety Using Working Memory Maintenance

Learn contextual and cued fear conditioning in mice using an automated video analysis system. This standardized protocol measures freezing behavior to assess fear learning and memory reliably.

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

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