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 border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr>
			<td>&#160;ImageFZ&#160; program</td>
			<td>Developed by Tsuyoshi Miyakawa</td>
			<td></td>
			<td>&#160;This program is available through O&#39;Hara &#38; Co., Tokyo, Japan and for free download at http://www.mouse-phenotype.org/. This software runs on 32-bit Windows XP/Vista/7.&#160;</td>
		</tr>
		<tr>
			<td>Conditioning chamber</td>
			<td>O&#39;Hara &#38; co., Japan</td>
			<td>CL-3002L</td>
			<td>For mouse.</td>
		</tr>
		<tr>
			<td>Cued test chamber</td>
			<td>O&#39;Hara &#38; co., Japan</td>
			<td>CLT-3002L</td>
			<td>For mouse.</td>
		</tr>
		<tr>
			<td>Interface</td>
			<td>O&#39;Hara &#38; 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&#39;Hara &#38; 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&#39;Hara &#38; 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&#39;Hara &#38; 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&#160; ( 2.5 cm spacing)</td>
		</tr>
	</tbody>
</table>

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 &#177; SD (g), 31.4&#177;3.55; mean body size &#177; SD (pixels), 351.6&#177;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="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50871/50871fig1highres.jpg" src="/files/ftp_upload/50871/50871fig1.jpg" /> 
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="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50871/50871fig2highres.jpg" src="/files/ftp_upload/50871/50871fig2.jpg" /> 
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="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50871/50871fig3highres.jpg" src="/files/ftp_upload/50871/50871fig3.jpg" /> 
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="Figure 4" fo:content-width="6in" src="/files/ftp_upload/50871/50871fig4highres.jpg" width="500" /> 
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="Figure 5" fo:content-width="6in" src="/files/ftp_upload/50871/50871fig5highres.jpg" width="500" /> 
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="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/50871/50871fig6highres.jpg" src="/files/ftp_upload/50871/50871fig6.jpg" /> 
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&#60;0.05; &#8224;, p&#60;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="Figure 7" fo:content-width="4.3in" src="/files/ftp_upload/50871/50871fig7highres.jpg" width="420" /> 
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&#60;0.0001</td>
			<td>F(7,56)=16.58, p&#60;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&#60;0.0001</td>
			<td>F(7,56)=11.69, p&#60;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&#60;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.

Watch this Scientific Journal Video about Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice at JoVE.com

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

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 ...

contextual-cued-fear-conditioning-test-using-video-analyzing-system

Research

JoVE Journal

Behavior

50.9K Views.  Fujita Health University. 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.

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

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 ...

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

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

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

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

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

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.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

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

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, sex, condition, and genetic background of the emitting animal. Although many studies have characterized these differences, whether receiver mice can discriminate among objectively different USVs and show preferences for particular sound traits remains to be elucidated. To determine whether mice can discriminate between different characteristics of USVs, a playback experiment was developed recently, in which preference responses of mice to two different USVs could be evaluated in the form of a place preference.
First, USVs from mice were recorded. Then, the recorded USVs were edited, trimmed accordingly, and exported as stereophonic sound files. Next, the USV amplitudes generated by the two ultrasound emitters used in the experiment were adjusted to the same sound pressure level. Nanocrystalline silicon thermo-acoustic emitters were used to play the USVs back. Finally, to investigate the preference of subject mice to selected USVs, pairs of two differing USV signals were played back simultaneously in a two-choice test box. By repeatedly entering a defined zone near an ultrasound emitter and searching the wire mesh in front of the emitter, the mouse reveals its preference for one sound over another. This model allows comparing the attractiveness of the various features of mouse USVs, in various contexts.

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.

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, ...

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse's head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.

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.

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive ...

Reducing State Anxiety Using Working Memory Maintenance

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working Memory (WM) and the threat of shock paradigms. In the Sternberg WM paradigm, subjects are required to maintain a series of letters in the WM for a brief interval and respond by identifying whether the position of a given letter in the series matches a numerical prompt. In the threat of shock paradigm, subjects are exposed to alternating blocks where they are either at risk of receiving unpredictable presentations of a mild electric shock or are safe from the shock. Anxiety is probed throughout the safe and threat blocks using the acoustic startle reflex, which is potentiated under threat (Anxiety-Potentiated Startle (APS)). By conducting the Sternberg WM paradigm during the threat of shock and probing the startle response during either the WM maintenance interval or the intertrial interval, it is possible to determine the effect of WM maintenance on APS.

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

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working ...

Using Electroencephalography Measurements and High-quality Video Recording for Analyzing Visual Perception of Media Content

This article explores a method to detect differences in visual perception in humans. The method used is based on the psychological (or "cognitive") function of eyeblinks. Participants' eyeblinks are detected and acquired while watching videos specifically created for the investigation. The detection and acquisition of eyeblinks are carried out with the help of a 20-channel electroencephalographic (EEG) wireless device. The international 10-20 system for electrode placement is followed. A high-definition (HD) video camera is used to record participants' facial expressions, for contrast purposes. Instead of using pre-existing media content, purpose-made video content has been created following specific criteria of interest for this investigation, with stimuli enabling researchers to manage the precise parameters of interest. Otherwise, results could be contaminated with uncontrolled variables. The synchronization of the presentation of video stimuli with EEG recordings needs to be done in milliseconds. Analysis of collected data is performed with robust software for working with big matrices. Statistically significant differences in eyeblink rate related to media professionalization and editing style are found with the reported experimental procedures.

We present detection, acquisition, and analysis of eyeblink rates while watching media content.

This article explores a method to detect differences in visual perception in humans. The method used is based on the psychological (or "cognitive") ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

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

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

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