Fear conditioning is, in short, a form of associative learning in which a fearful response is associated with a stimulus or location. After pairing the stimulus/location with an aversive stimulus, subjects exhibit fearful responses (e.g. freezing behavior in rodents) in response to the location in the absence of the previously paired aversive stimulus. However, when the animal is subsequently exposed to this location repeatedly without experiencing the negative stimulus, this association eventually fades in a process known as extinction. This process has been championed as a measure of cognitive plasticity, the brain’s ability to adapt to change. Further, extinction protocols may provide key insights into approaches for the treatment of such chronic conditions as post-traumatic stress disorder, in which previous pairings of otherwise neutral things and places become strongly linked with negative experiences, leading to ongoing mental distress.
As with fear conditioning itself, extinction is an extremely well-conserved feature of memory across species. Extinction was first observed by Pavlov in his seminal experiments with canines. After training the dogs to salivate in response to a sound which had previously been paired with appetitive food, the dogs slowly lost this paired response over repeated trials in which the sound was presented in the absence of food. Nearly 80 years later, protocols were developed to investigate this very phenomenon in humans using the startle reflex as well as virtual reality paradigms. Much of the neurological basis for fear conditioning and extinction which had been established in rodents and other mammals was confirmed in humans using PET and fMRI imaging.[6,7]
What Brain Regions Are Involved in Fear Conditioning Extinction?
The earliest work acting to unveil the neurological mechanisms underlying extinction were championed by Falls and colleagues, who showed that NMDA receptors in the basolateral amygdala (BLA) were required for this process. Established knowledge indicating that the BLA is a key driver of fear conditioning and other forms of contextual and emotional learning, paired with the fact that NMDA receptors are important mechanistic drivers of synaptic plasticity, led this to be a quickly accepted understanding of the extinction process. However, further experiments quickly revealed that the circuitry underlying extinction is substantially more complex.
A 1993 study by Morgan et al was the first to surmise that, based on strong connectivity between the amygdala (including the BLA) and the medial prefrontal cortex (mPFC), the BLA-mPFC pathway is a crucial component of the extinction process. While lesions of the mPFC were not found to prevent the acquisition of the fear response during the initial fear conditioning experiments, they effectively prevented the extinction process. Many further studies confirmed the role of the mPFC in extinction using lesion, drug inactivation, and regional stimulation and further refined these findings to implicate a subregion of the mPFC in rodents known as the infralimbic cortex (IL).[10-12] In humans, the IL equivalent is known as the ventromedial prefrontal cortex.
As with nearly all forms of learning, extinction also requires the participation of the hippocampus. Two seminal papers from 1989 solidified this idea through lesions of sensory cortices which directly interfaced with both the amygdala and hippocampus.[14,15] Based on the knowledge that the ventral hippocampus exhibits reciprocal connectivity with both the BLA and the mPFC, innumerable rodent studies have confirmed these findings. Thus, while several other brain regions may be involved in some capacity, the primary circuitry underlying extinction processing is agreed to include the BLA, the mPFC and the hippocampus.
Fear Conditioning Extinction Protocol (context discrimination)
- Ensure that your testing apparatus is clean (free from any residual odors or tactile cues such as leftover bedding). This can be achieved by wiping all surfaces with a 70% EtOH solution and allowing these surfaces to dry completely.
- Habituate your animal to the testing room for a minimum of 60 minutes prior to introducing them to the apparatus.
- Introduce the animal to the paired context previously used for the induction of a conditioned fear response.
- After 3 minutes in the context, present the conditioned stimulus (ex. auditory/tone; 10 seconds; 80dB) without the unconditioned stimulus. Repeat this presentation 30 times with 70 second intervals.
- Following the final (30th) conditioned stimulus presentation, remove the animal from the context within 60 seconds and return the animal to their home cage.
- 24 hours after Step 5, proceed to repeat Step 1-5 in order to test for retention or extinction of the fear response.
Fear conditioning extinction is a simple task used to measure cognitive plasticity of paired memories which had previously been induced by cued or contextual fear conditioning. This form of learning is crucial to understanding the brain’s ability to adapt to changing environments and conditions, and may be key to the conceptualization of therapeutic approaches for the treatment of such conditions as PTSD and chronic anxiety disorders. Protocols for fear conditioning extinction are a direct extension of the acquisition protocols and are a simple, reliable way of probing memory and plasticity functions in rodents.
- Chang, C., Knapska, E., Orsini, C. A., Rabinak, C. A., Zimmerman, J. M., & Maren, S. (2009). Fear Extinction in Rodents. Current Protocols in Neuroscience / Editorial Board, Jacqueline N. Crawley … [et Al.], CHAPTER, Unit8.23.
- Maren, S., Phan, K. L., & Liberzon, I. (2013). The contextual brain: Implications for fear conditioning, extinction and psychopathology. Nature Reviews. Neuroscience, 14(6), 417–428.
- Pavlov, I. (1927). Conditioned Reflexes. Oxford University Press.
- Jovanovic, T., Keyes, M., Fiallos, A., Myers, K. M., Davis, M., & Duncan, E. J. (2005). Fear potentiation and fear inhibition in a human fear-potentiated startle paradigm. Biological Psychiatry, 57(12), 1559–1564.
- Baas, J. M., Nugent, M., Lissek, S., Pine, D. S., & Grillon, C. (2004). Fear conditioning in virtual reality contexts: A new tool for the study of anxiety. Biological Psychiatry, 55(11), 1056–1060.
- Semple, W. E., Goyer, P. F., McCormick, R., Compton-Toth, B., Morris, E., Donovan, B., … Schulz, S. C. (1996). Attention and regional cerebral blood flow in posttraumatic stress disorder patients with substance abuse histories. Psychiatry Research, 67(1), 17–28.
- Shin, L. M., McNally, R. J., Kosslyn, S. M., Thompson, W. L., Rauch, S. L., Alpert, N. M., … Pitman, R. K. (1999). Regional cerebral blood flow during script-driven imagery in childhood sexual abuse-related PTSD: A PET investigation. The American Journal of Psychiatry, 156(4), 575–584.
- Falls, W. A., Miserendino, M. J., & Davis, M. (1992). Extinction of fear-potentiated startle: Blockade by infusion of an NMDA antagonist into the amygdala. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 12(3), 854–863.
- Morgan, M. A., Romanski, L. M., & LeDoux, J. E. (1993). Extinction of emotional learning: Contribution of medial prefrontal cortex. Neuroscience Letters, 163(1), 109–113.
- Akirav, I., & Maroun, M. (2007). The Role of the Medial Prefrontal Cortex-Amygdala Circuit in Stress Effects on the Extinction of Fear. Neural Plasticity, 2007.
- Quirk, G. J., Likhtik, E., Pelletier, J. G., & Paré, D. (2003). Stimulation of medial prefrontal cortex decreases the responsiveness of central amygdala output neurons. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 23(25), 8800–8807.
- Holmes, A., & Wellman, C. L. (2009). Stress-induced prefrontal reorganization and executive dysfunction in rodents. Neuroscience and Biobehavioral Reviews, 33(6), 773–783.
- Phelps, E. A., Delgado, M. R., Nearing, K. I., & LeDoux, J. E. (2004). Extinction learning in humans: Role of the amygdala and vmPFC. Neuron, 43(6), 897–905.
- Ledoux, J. E., Romanski, L., & Xagoraris, A. (1989). Indelibility of subcortical emotional memories. Journal of Cognitive Neuroscience, 1(3), 238–243.
- Teich, A. H., McCabe, P. M., Gentile, C. C., Schneiderman, L. S., Winters, R. W., Liskowsky, D. R., & Schneiderman, N. (1989). Auditory cortex lesions prevent the extinction of Pavlovian differential heart rate conditioning to tonal stimuli in rabbits. Brain Research, 480(1–2), 210–218.
- Hugues, S., & Garcia, R. (2007). Reorganization of learning-associated prefrontal synaptic plasticity between the recall of recent and remote fear extinction memory. Learning & Memory, 14(8), 520–524.