Name: fear incubation using an extended fear-conditioning protocol for rats
Uploaded: 2020-08-22T13:00:00
Description: Watch this Scientific Journal Video about Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats at JoVE.com

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

The following protocol&#160;was&#160;approved by the Institutional Animal Care and Use Committee of Fundaci&#243;n Universitaria Konrad Lorenz (IACUC-KL). The universal declaration of animal rights issued by International League of Animal Rights, Geneva, Switzerland (1989), and ethical principles of experimentation with animals issued by ICLAS were respected.
1. Subject preparation
<ol>
	<li>Select male adult Wistar rats (n = 12). House them in groups of four per cage for three days of accli...

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

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

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fear incubation using an extended fear-conditioning protocol for rats

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

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

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

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats (Video) | JoVE

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

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

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