Fear Conditioning

Cued Fear Conditioning

By June 5, 2019 June 8th, 2019 No Comments

Cued fear conditioning is a form of associative learning[1] in which an animal is trained to associate a cue (often a sound) with a fear-inducing stimulation (such as a foot shock or an air puff). Associative learning models such as fear conditioning are dictated by two stimuli, the unconditioned stimulus, and the conditioned stimulus. A conditioned stimulus is a neutral signal such as a tone, while the unconditioned stimulus carries a valence (in this case negative) that becomes associated with the previously neutral conditioned stimulus. Importantly, this form of fear conditioning often overlaps with context fear conditioning in that, obligatorily, the unconditioned stimulus presentation must occur in a context of some sort. In order to separate these two forms of fear conditioning, the subject should be pre-habituated to the context (i.e. behavioral arena) in which the conditioning will eventually take place. In this way, the subject associates the context with cognitive and emotional states other than the fear conditioning protocol.

Within cued fear conditioning there are two primary models, delay conditioning and trace conditioning. Delay conditioning occurs when the unconditioned stimulus is presented either immediately after or in conjunction with the conditioned stimulus. Normally, this is achieved by presenting the cue (tone, light or odor) for a given duration at the same time as the aversive stimulus, or with the stimulus occurring at the end of the cue (in the case of a tone or light). Conversely, trace conditioning refers to the model in which the unconditioned stimulus is presented following a time delay (“trace”) after the conditioned stimulus. In this case, the cue is terminated and the aversive stimulus is given following a trace of between 100ms and one minute.

What brain regions are involved in cued fear conditioning?

Understanding the brain regions involved in cued fear conditioning is the first step towards knowing what conditions, disorders, and drugs might impact behavior in this task. A number of brain regions have been noted as contributing to cued fear conditioning with some differences regarding the type of cue and the type of response.

First, the amygdala has been shown to mediate significant aspects of cued delay-type fear conditioning.[2] Indeed, the basolateral amygdala is the region in which the association between the conditioned and unconditioned stimulus is solidified[3] via a protein-synthesis dependent mechanism.[4] The expression of fear behaviors is then mediated by signals from the basolateral amygdala[5] to the central nucleus of the amygdala.[6] Obviously, the amygdala is an important structure in delay fear conditioning. Additionally, two subregions of the hippocampus known as CA3 and the dentate gyrus have been shown to be activated following delay fear condition.[7] While parts of the hippocampus are involved in both delay and trace fear conditioning, these two subregions are preferentially activated by the delay-type experiments.

Trace fear conditioning requires contributions from other brain regions and is, therefore, an interesting tool for investigating disorders which involve these areas. Early studies have shown that the hippocampus is involved in auditory trace-conditioning, as exemplified by a loss of this behavioral association in animals where the hippocampus has been inactivated.[8] In addition to this, pharmacological blockade of GABA or NMDA receptors in the prefrontal cortex also inhibits the formation of trace-type fear conditioning, indicating that this region is also required for this behavior.[9] Interestingly, the basal amygdala, while not involved in delay-type fear conditioning, is required for trace fear conditioning.[2] Similarly, the hippocampus plays an important role in trace conditioning that is less relevant for delay conditioning. In rodents and humans, the hippocampus is increasingly important when the delay between the unconditioned and conditioned stimulus is increased, indicating a particular role in the cue association which occurs following this time gap.[10]

Cued Fear Conditioning Protocol

  1. Ensure that your testing apparatus is clean (free from any residual odors or tactile cues such as leftover bedding).
  2. Habituate your animal to the testing room for a minimum of 60 minutes prior to introducing them to the apparatus.
  3. Habituate your animal to the apparatus to ensure that they do not associate the environment with the fear-inducing stimulus. This can be done with a short (~5min) session the day prior to the fear conditioning or immediately prior to the fear conditioning session.
  4. Present the conditioned stimulus (example: a 70-80dB tone) for 30 seconds.
  5. For delay-conditioning, present the unconditioned stimulus (example: a footshock of 0.6mA) for a duration of 1-2 seconds during the last 1-2 seconds of the conditioned stimulus. For trace-conditioning, insert a pause of 2-60 seconds following the conditioned stimulus before the presentation of the unconditioned stimulus.
  6. Remove the animal within 30-60 seconds of the unconditioned stimulus in order to prevent context-association.
  7. On day 2, repeat steps 1-6.


Cued fear conditioning is a simple test of learning based on pairing a neutral (conditioned) stimulus with an aversive (unconditioned) stimulus in order to elicit a fear response. This technique can be done either by an immediate pairing (delay conditioning) or with a time delay between the stimuli (trace conditioning). While seemingly similar, these two forms of cued fear conditioning involve distinct brain regions and are therefore impacted by different disorders and conditions. In both cases, the protocol is simple to execute and provides a reliable measure of emotional memory.


  1. Kim JJ, Jung MW. Neural circuits and mechanisms involved in Pavlovian fear conditioning: A critical review. Neurosci. Biobehav. Rev. 2006;30(2):188.
  2. Kochli, D. E., Thompson, E. C., Fricke, E. A., Postle, A. F., & Quinn, J. J. (2015). The amygdala is critical for trace, delay, and contextual fear conditioning. Learning & Memory, 22(2), 92–100.
  3. Barot, S. K., Chung, A., Kim, J. J., & Bernstein, I. L. (2009). Functional imaging of stimulus convergence in amygdalar neurons during Pavlovian fear conditioning. PloS One, 4(7), e6156.
  4. Kwapis, J. L., Jarome, T. J., Schiff, J. C., & Helmstetter, F. J. (2011). Memory consolidation in both trace and delay fear conditioning is disrupted by intra-amygdala infusion of the protein synthesis inhibitor anisomycin. Learning & Memory (Cold Spring Harbor, N.Y.), 18(11), 728–732.
  5. LeDoux, J. E. (1993). Emotional memory systems in the brain. Behavioural Brain Research, 58(1–2), 69–79.
  6. LeDoux JE, Iwata J, Cicchetti P, Reis DJ 1988. Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear. J Neurosci 8: 2517–2529.
  7. Weitemier, A. Z., & Ryabinin, A. E. (2004). Subregion-specific differences in hippocampal activity between Delay and Trace fear conditioning: an immunohistochemical analysis. Brain Research, 995(1), 55–65.
  8. McEchron MD, Bouwmeester H, Tseng W, Weiss C, Disterhoft JF 1998. Hippocampectomy disrupts auditory trace fear conditioning and contextual fear conditioning in the rat. Hippocampus 8: 638–646.
  9. Gilmartin MR, Helmstetter FJ 2010. Trace and contextual fear conditioning require neural activity and NMDA receptor-dependent transmission in the medial prefrontal cortex. Learn Mem 17: 289–296.
  10. Clark RE, Squire LR. Classical conditioning and brain systems: The role of awareness. Science. 1998;280(5360):77–81.

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.