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

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.