Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

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

Memory is a psychological process that entails various phases information acquisition, consolidation and retrieval. During the consolidation phase, establishment of new synaptic connections and modification of pre-existing connections occurs. This phase of consolidation depends on the value of the stimuli with a stronger memory trace when the stimuli are emotionally relevant.

The majority of the studies on emotional memory have focused on the amygdala's nucleus and the relation with other brain structures. This phenomena having primarily studied with fear condition paraments. Fear condition is a form of learning which enables learning the relationships between aversive events and otherwise neutral stimuli.

One of the most widely used procedures is classical or Pavlovian conditioning in mammals. Including rodents and humans. In the case of rodents, the conditions symbols or US is typically a picture which is presented a single or several times across a single or several sessions.

Fear conditioning protocols typically entail one to two ton shocks vary. However, other less used aversion involve extensive not burned bearings. This is called averse training which results in a long lasting memory effect called a fear incubation.

Fear incubation entails the increase of the intensity of conditionate response over time. These in the upset of further exposure to other such event or conditionate stimuli. Recent understanding of fear incubation in behavioral and neurobiological nerves has shown isolation to other rodents and psychological phenomena including delayed answer for certain disorders Here we describe an extending fear condition in prime in which we tested contextual and Cue-memory for 48 hours and six weeks after and over 20 sessions.

This allows us to test the strength of the emotional memory over time. Select 12 male, adult Wistar Rats and house them in groups of four in a cage, with free access to water throughout the experiment. Maintain the rats at 85%of their free feeding weight, giving restricted food on the same day, hour every day.

Randomly assign subjects to the following groups:Emotional testing, six week after training. Emotional testing, 48 hours after training. Clean all the internal surfaces of the experimental chamber and grid floor with 10%ethanol solution.

Connect the red and black clips on the shock intensity calibrator to two rods on the grid floor. Connect the USB cable to the corresponding port of the computer. Start the shock intensity calibrator software.

Choose one milliamp intensity in the application by clicking on the button. Then change the run/stop switch to run. Operate the aversive stimulator and look at the shock intensity displayed on the panel of the application.

If needed adjust the intensity to one milliamp using the knob on the aversive stimulator. Close the experimental chamber and sound out the narrating box. Start the freezing detection system software and open the experimental dialogue window.

Enter the details of each subject and load the training protocol file. Choose the corresponding camera and check the side video option. Set the motion threshold to 100 and minimal freeze ratio to 13 frames.

Verify the live feed from the chosen camera appears on the screen. Together with the the motion threshold graph and the timeline of the different stimuli that are presented during the training. Click the calibrate option three times while checking that the motion index remains below 100 Then select equipment to lock by clicking on the corresponding button on the screen.

Introduce the experimental subject to the chamber. Start the session by clicking on the record button, 25 tone shock pairings or trials are delivered with a 60 second inter trial interval. Starting on the minutary of the session.

The tone is presented during the last 10 second of each inter trial interval and the shock during the last two second of each inter trial interval. Remove the rat from the experimental chamber when the 28 minute session is over. Return animals to their respective home cage.

Transport the rat in their home cages cupboard. From the behavioral training room to the animal care facility. Rats remain 48 hours or six weeks without any treatment depending on which group they were assigned.

During this period the animals rest in their home cages and are housed individually. Monitor the weight of the animals twice per week during the six weeks of the incubation period. Gently, manipulate each animal for two minutes while they're weighted.

Six animals may be tested 48 hours after timing. Those six animals must be tested six weeks later. Repeat freezing detection system calibration.

Load the file name, contextual test protocol. Start the session by clicking on the record button. During this single 10 minute context test session, no stimuli are presented.

One day after the context test, repeat the following steps with each animal, depending on the group. To change the visual context, a plastic surrounding wall was inserted to the experimental chamber. To change the olfactory context, one percent acetic acid was applied to our cotton taped swab.

Which was placed in the metal tray, below the grid floor. Repeat freezing detection system calibration using a different file. Load the file named Cue test protocol'Start the session by clicking on the record button.

During this single 15 minute Cue test session, the conditionate stimulus tone is presented 10 times starting on minute three of the session. That is 10 Cue test trials and the reboot with the same inter trial inter walls in absent of shocks. Use additional custom made programming to obtain:A percentage of freezing to the tones during the training and Cue-test sessions.

B percentage of freezing for each of eight three-min bins of the training session. C percentage of freezing during the first three mins of the training session. Consider this last measure as baseline freezing since no shocks or bones were presented during that three minute period.

To obtain this data open freezing detection system software and follow these steps. Select file, reports, bench components summary. Select the cmd file available on the free repository Open Science Framework'Name then open file.

Change the motion threshold to 100 and click okay Select files to be analyzed. Open files can be opened in Microsoft excel. For further analysis.

Lastly, calculate the average duration of each freezing episode. For each session by dividing the total freezing duration over the total number of freezing episodes. Animals were active and explored the experimental chamber during the three minute baseline period.

Baseline freezing time was significantly lower when compared to the remaining of the session. This is an indication of fear acquisition. Segmenting the trial session in three minute bins shows that the percentage of time allocated to freezing reached an asymptote during the first three shock trials.

This finding has been consistent in previous research and indication of other training. Percentage of freezing of all animals during the training period was significantly higher than the baseline period. Animals tested six weeks after training showed significantly higher percentages of freezing during the context test than animals tested at 48 hours.

No differences in freezing time were observed between training and memory tests. The freezing response of the different groups of subjects during the context test was further explored with other measures. The activity level of subjects that were tested six weeks after training was significantly lower than that of animals tested 48 hours after the conditioning section.

Freezing times of the animals tested shortly after training, was significantly lower than that of animals tested six weeks after. These results indicate a fear incubation effect. An analysis of the averse reaction of each freezing episode indicated that animals tested 48 hours of their conditioning session displayed longer freezing episodes than animals tested 48 hours after training.

Freezing percentages of all animals during baseline of training session and Cue test was significantly lower than during the test period of Cue test. Only baseline of the animal tested six weeks later in the Cue test was significantly different from the test period of Cue test. Mean freezing during Cue training was significantly lower than the mean freezing during Cue test after six weeks.

Mean freezing during inter trial interval in Cue test was significantly lower than the mean freezing during inter trial interval in training after 48 hours. Here we describe an extended fear conditioning protocol that produced over training in fear incubation rat. It is an efficient and valid approach to assess emotional memory across short 48 hours and long term periods six weeks.

There is a mountain pharmacological, physiological and anatomical evidence that supports its validity for assessing emotional memory phenomena. Among the different advantages of this protocol are the following:First, the first two types of memory test, namely contextual and Cue. This test allows to identify the differential effect of the testing the line for context and Cue.

Second, requires a single 28 minute training session which in turn produces a long term effect that goes beyond several weeks. This advantage is remarkable considering that some person expected fear conditioning needs at least 100 shocks across 10 sessions of training. Fear offers several measurement alternatives which are calculated automatically.

Compared to other fear conditioning paradigms with brief training sessions. Extended protocols that produce averse training effects have received less attention. A relevant effect associated with such averse training is fear incubation which only recently began to be understood in a behavioral and neurobiological level.

And seems related to other phenomena, including delayed onset of post traumatic stress disorder. Final recommendations for the best implementations and research of these protocols includes:correct cleaning of the experimental chamber, especially the grip floor. Calibration, the shock intensity prior to turning the shock shock on and freezing detection system calibration.

This protocol could be tested with all breeds of rats or other rodents. In those cases it is important to adjust the motion threshold and shock intensity.