Fear Conditioning

Contextual Fear Conditioning

By May 30, 2019 June 8th, 2019 No Comments

Contextual fear conditioning is an associative learning test in which a test subject, most often a mouse or rat, learns to associate an environment (context) with a fear-inducing stimulus.[1] As with other forms of associative learning, such as cued fear conditioning, this form of learning is dictated by a neutral stimulus (termed “conditioned stimulus”) and a valence-associated stimulus (termed “unconditioned stimulus”). Contextual fear conditioning, however, may also be performed with the context itself acting as the conditioned stimulus, rather than a presented stimulus such as a tone. Most commonly, contextual fear conditioning is achieved through the use of a foot shock as the unconditioned stimulus,[2] which the animal learns to associate with a novel environment. Unlike with cued fear conditioning, no variations in the timing of acquisition protocols are used with contextual fear conditioning, since the absence of a manipulated conditioned stimulus implies that no such modification can be made.

Contextual fear conditioning is a remarkably consistent feature of learning that has been conserved throughout evolution, due to its evident utility in natural environments.[3] In essence, all animals must learn to associate certain environments with the threat of danger in order to survive. Thus, when an environment is paired with an aversive stimulus, animals exhibit defensive mechanisms which, in rodents, is primarily the freezing reflex. As a translational model, contextual fear conditioning has been used both as a simple means of understanding the mechanisms underlying memory formation and recall, as well as a basis for studying human-centric disorders such as post-traumatic stress disorder (PTSD).[4]

The most basic variant of contextual fear conditioning is known as the one-trial procedure, in which an animal is placed into a novel environment, after which an aversive stimulus is delivered. The animal may then be tested for context-dependent fear responding at a variety of intervals following this procedure in order to assess the strength and duration of the memory. In addition to this basic model, a number of important variations on contextual fear conditioning exist, including (but not limited to): context pre-exposure, context discrimination, and immediate shock.[5]

What Brain Regions Are Involved in Contextual Fear Conditioning?

A basic understanding of the neurological correlates of contextual fear conditioning provides the basis for understanding what potential disorders, diseases or drugs may affect behavior in the task. While many brain regions implicated in other forms of learning, such as cued fear conditioning, are similarly implicated in contextual fear conditioning, some important differences have been noted.

Most prominently, the amygdala appears to play a particularly important role in the learning and recall of contextual fear conditioning. Indeed, the three major subregions of the amygdala (lateral, basal and central) have all been identified as crucial for this learning model.[6] While lesion studies have confirmed the importance of the amygdala in multiple forms of fear conditioning, including contextual, there is some debate as to whether or not the basolateral amygdala is crucial for this type of association.[7]

In addition to this amygdalar participation in contextual fear conditioning, the hippocampus plays a crucial role in any form of learning involving context. Indeed, the hippocampus is defined as the brain region most important for understanding space and place, in particular, the dorsal hippocampus and the CA3 region, wherein cellular fields are comprised of “place cells” which are activated in specific contexts.[8] This implies that any context association requires the participation of these hippocampal regions, a finding similarly shown in humans with hippocampal damage.[9]

Finally, the prefrontal cortex has been shown to participate heavily in the processing of contextual fear conditioning. The prefrontal cortex has previously been shown to participate in a variety of functions crucial to such a learning experience, including emotional regulation, threat evaluation, and context-encoding.[10] Given these important roles, it is not surprising that alterations in prefrontal cortex function induce significant changes in contextual fear conditioning behavior.[11]

Contextual Fear Conditioning Protocol (one-trial)

  1. 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.
  2. Habituate your animal to the testing room for a minimum of 60 minutes prior to introducing them to the apparatus.
  3. Introduce the animal into the testing context for 2 minutes with no additional stimuli.
  4. If using a conditioned stimulus (such as a tone) as well, present the tone for 30 seconds. If not, proceed to step 5.
  5. Deliver the unconditioned stimulus (example: a footshock of 0.6mA) for a duration of 1-2 seconds (if using a conditioned stimulus, do this during the last 1-2 seconds of the tone, for example).
  6. If using multiple pairings, repeat steps 3-5.
  7. Remove the animal within 30-60 seconds of the conditioned stimulus.
  8. Testing of the fear response (e.g. freezing behavior) may be commenced on day 2. To test for the freezing response, place the animal in the context from day 1 and note whether the animal is moving or not (i.e. frozen, meaning the absence of any movement aside from respiration) once every 10 seconds over the course of a 5 minute period.

Contextual Fear Conditioning Protocol (pre-exposure)

Prior to commencing step 1 in the above protocol, the animal should be exposed to the context for a brief duration. Some animals have been shown to exhibit enhanced contextual fear conditioning acquisition if given a contextual pre-exposure.[12] This pre-exposure should last between 30 seconds and several minutes and must take place in identical conditions to the above protocol (i.e. the same apparatus, cleaned and prepared as in step 1).

Contextual Fear Conditioning Protocol (context discrimination)

For context discrimination, animals are exposed to two contexts with differing cues (such as visual, olfactory or auditory signs which differ between the two contexts).[13] This protocol has been suggested to enhance hippocampal participation in context association as the animal must differ between two related contexts, one which is paired with an aversive stimulus and another which is not.

  1. On the day prior to the contextual fear conditioning protocol (i.e. delivery of an aversive stimulus in the associated context), the animal should be exposed to two environments (A and B) which differ by as many cues as possible (visual cues and olfactory cues) and must be located in two different testing rooms. As before, the environments must be cleaned prior to use with 70% EtOH and allowed to dry fully before testing begins. The animal should be placed in each of the two contexts for 5 minutes with one hour between the two exposures.
  2. On the second day, the animal is exposed to the contextual fear conditioning protocol described above in one of the two contexts (A or B).
  3. Following the contextual pairing, return the animal to the holding cage for one hour.
  4. Place the animal into the non-paired context (i.e. if the fear conditioning took place in context A, now you will place them into context B) for an equivalent period of time.
  5. Repeat steps 2-4 for a total of 4 days.
  6. On day 5, test the animal for the freezing response in both contexts (with one hour between the two exposures) and compare freezing times between the paired and unpaired environment.

Protocol note: In addition to control animals who receive no aversive stimulus (i.e. those who are exposed to the context without ever receiving a footshock), some researchers use the “immediate shock” protocol as an additional control. In order to perform this control, skip steps 3-4 of the basic contextual fear conditioning protocol. In other words, the aversive stimuli should be delivered immediately when the animal is introduced to the context. In this way, the animal does not have time to associate the context with the shock. Thus, this control can be used to eliminate effects seen as a result of the shock itself, rather than effects that result from a contextual association to the shock.[14]


Contextual fear conditioning is a relatively simple procedure which can be used to measure associative learning. This form of learning involves multiple brain regions, including the amygdala, the hippocampus, and the prefrontal cortex. No pre-training is required (except in the case of pre-exposure or context discrimination protocols), making contextual fear conditioning a quick protocol for measuring associative learning.


  1. Rustay, N., Browman, K., & Curzon, P. (2008). Cued and Contextual Fear Conditioning for Rodents. Frontiers in Neuroscience, 19–37.
  2. Curzon P, Rustay NR, Browman KE. Cued and Contextual Fear Conditioning for Rodents. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009.
  3. LeDoux, J.E. 1994. Emotion, memory and the brain. Sci. Am. 50-57.
  4. Layton, B. and Krikorian, R. 2002. Memory mechanisms in posttraumatic stress disorder. J. Neuropsychiatry Clin. Neurosci. 14:254-261.
  5. Wehner, J. M., & Radcliffe, R. A. (2004). Cued and Contextual Fear Conditioning in Mice. Current Protocols in Neuroscience.
  6. Goosens KA, Maren S. Contextual and auditory fear conditioning are mediated by the lateral, basal, and central amygdaloid nuclei in rats. Learn. Mem. 2001;8(3):148–155.
  7. Vazdarjanova A, McGaugh JL. Basolateral amygdala is not critical for cognitive memory of contextual fear conditioning. Proc. Natl. Acad. Sci. USA. 1998;95(25):15,003–15,007.
  8. Muller, R. U.; Kubie, J. L. (1987). “The effects of changes in the environment on the spatial firing of hippocampal complex-spike cells”. The Journal of Neuroscience. 7 (7): 1951–68.
  9. Clark RE, Squire LR. Classical conditioning and brain systems: The role of awareness. Science. 1998;280(5360):77–81.
  10. Rozeske, R. R., Valerio, S., Chaudun, F., & Herry, C. (2014). Prefrontal neuronal circuits of contextual fear conditioning. Genes, Brain and Behavior, 14(1), 22–36.
  11. Gilmartin, M. R., & Helmstetter, F. J. (2010). Trace and contextual fear conditioning require neural activity and NMDA receptor-dependent transmission in the medial prefrontal cortex. Learning & Memory, 17(6), 289–296.
  12. Rudy, J.W., Barrientos, R.M., and O’Reilly, R.C. 2002. Hippocampal formation supports conditioning to memory of a context. Behav. Neurosci. 116:530-538.
  13. Frankland, P.W., Cestari, V., Filipkowski, R.K., McDonald, R.J., and Silva, A.J. 1998. The dorsal hippocampus is essential for context discrimination but not for contextual conditioning. Behav. Neurosci. 112:863-874.
  14. Young, E.A., Owen, E.H., Meiri, K.F., and Wehner, J.M. 2000. Alterations in hippocampal GAP-43 phosphorylation and protein level following contextual fear conditioning. Brain Res. 860:95- 103.

About Andrew Scheyer

Andrew Scheyer is a post-doctoral fellow at the Institut de Neurobiologie de la Méditerranée. He completed his PhD in Neuroscience in 2015 at Rosalind Franklin University of Medicine and Science, studying the synaptic underpinnings of protracted withdrawal from cocaine self-administration. Currently, his research focuses on the impact of marijuana exposure on neurological development. He specializes in electrophysiological studies of synaptic plasticity and in vivo deep-brain calcium imaging in freely moving rats.