Contingency awareness shapes neural responses in fear conditioning

Investigating the impact of sex and cortisol on implicit fear conditioning with fMRI

Understanding what an animal learns when exposed to novelty is of great interest to behavioral neuroscientists, but it can be challenging to understand what information is acquired in a particular learning session. The behavior of an animal has to be quantified using either visual or mechanical measures of a particular response. One way of elucidating mechanisms involved in discrete learning sessions is to study associative learning processes. Simplistically, associative learning is an adaptive process that allows an organism to learn to anticipate events.One form of associative learning that has been used in multiple species, including humans, is eye-blink conditioning. The most common species used, the rabbit, has yielded interesting results, especially in identifying and elucidating the involvement of the cerebral cortex. Similar procedures have been used in cats, rats, and humans. Another form of associative learning that has gained popularity with behavioral pharmacologists is fear conditioning. While the eye-blink procedure has overlap with context/cue fear conditioning and in many cases yields similar results, there are some basic differences between fear conditioning and eye-blink conditioning. One main difference is that eye-blink conditioning takes many more training trials to establish. Fear conditioning has gained popularity, in large part as a result of the need to characterize mutant mice and the effects of genetic alterations; therefore, this chapter primarily focuses on fear conditioning.Fear conditioning to either a cue or a context represents a form of associative learning that has been well used in many species [1]. The majority of the experiments reported in the literature involve the mouse; however, there is also a generous proportion of the literature devoted to the rat. There are also several reports in higher species that are not covered in this chapter. In general any of the procedures described in this chapter can be used for either the rat or the mouse.The dependent measure used in contextual and cued (delay or trace) fear conditioning is a freezing response that takes place following pairing of an unconditioned stimulus (US), such as foot shock or air puff, with a conditioned stimulus (CS), a particular context and/or such a cue. In the case of rats and mice, this US is generally a foot shock. Obviously, if in a conditioning context one administers a foot shock that is paired with a tone, there will be learning not only to the tone, but also to the context. Two types of conditioning that are typically employed are delay or trace conditioning. Delay conditioning refers to a situation in which the US is administered to co-terminate with or occur immediately after the CS. Trace conditioning differs from delay conditioning in that the US follows an empty (“trace”) interval that separates the cessation of the CS from the onset of the US. Trace conditioning adds additional complexity to delay conditioning, as the time interval between the CS and US requires the formation of a temporal relationship between the two stimuli.In this chapter we discuss the various challenges inherent in this type of procedure in order to enable the experimenter to set the conditions to best answer the questions being posed. One of the biggest advantages of cued and contextual fear conditioning in the rodent is that they are forms of passive learning that can be used in many strains of mice and rats, even when more pronounced motor deficits are problematic in other learning assays. As a consequence of these procedural advantages, contextual fear conditioning is gaining popularity, especially in the phenotyping of transgenic mice.

In an fMRI study, effects of contingency awareness on conditioned responses were assessed in three groups comprising 118 subjects. A differential fear-conditioning paradigm with visual conditioned stimuli, an electrical unconditioned stimulus and two distractors was applied. The instructed aware group was informed about the contingencies, whereas the distractors prevented contingency detection in the unaware group. The third group (learned aware) was not informed about the contingencies, but learned them despite the distractors. Main effects of contingency awareness on conditioned responses emerged in several brain structures. Post hoc tests revealed differential dorsal anterior cingulate, insula and ventral striatum responses in aware conditioning only, whereas the amygdala was activated independent of contingency awareness. Differential responses of the hippocampus were specifically observed in learned aware subjects, indicating a role in the development of contingency awareness. The orbitofrontal cortex showed varying response patterns: lateral structures showed higher responses in instructed aware than unaware subjects, the opposite was true for medial parts. Conditioned subjective and electrodermal responses emerged only in the two aware groups. These results confirm the independence of conditioned amygdala responses from contingency awareness and indicate specific neural circuits for different aspects of fear acquisition in unaware, learned aware and instructed aware subjects.

Classical fear conditioning involves pairing a neutral conditional stimulus (CS) with an aversive unconditional stimulus (US). Subsequent presentation of the CS alone induces fear responses. Acquisition of conditioned fear is thought to involve learning of the CS/US association, followed by memory consolidation. Recently we reported that the N1/P2 auditory evoked potential was enhanced by fear conditioning in humans. Diazepam 10 mg, given before CS/US pairing, prevented subsequent expression of fear potentiation when the response was elicited, 1 week later, in the presence of the CS. In this experiment, we examined whether this effect of diazepam was caused by disruption of the formation of CS/US associations or by disruption of consolidation. The benzodiazepine antagonist flumazenil was used to block the effect of diazepam either during the association period or during subsequent consolidation. Forty-two male volunteers (18-35 years) participated in two sessions separated by 7 days. In Session One, they ingested diazepam 10 mg or placebo: 60 minutes later they received flumazenil 1 mg or saline intravenously (i.v.). Then they received 20 presentations of a light (CS), 50% of which terminated with electric shock (US). This was followed by a second infusion of flumazenil or saline. Subjects received placebo/saline/saline (Group 1), diazepam/saline/saline (Group 2), diazepam/flumazenil/saline (Group 3) and diazepam/saline/flumazenil (Group 4). In Session Two, the CS was presented without the US; 50% of CS presentations terminated with a sound pulse; an equal number of sound pulses were presented without the CS. Auditory evoked potentials were recorded at Cz. In Session Two, CS presentation enhanced the auditory N1/P2 potential in placebo-treated subjects (Group 1). This enhancement was prevented by diazepam (Group 2). Flumazenil reversed diazepam's effect on fear potentiation if it was administered before conditioning (Group 3), but not if it was administered afterwards (Group 4). The results confirm that diazepam prevents the acquisition of fear conditioning in humans, and suggest that it disrupts the formation of CS/US associations, rather than the consolidation of fear memory.

Memory often combines information from different sensory modalities. Animal studies show that synchronized neuronal activity in the theta band (4–8 Hz) binds multimodal associations. Studies with human participants have likewise established that theta-phase synchronization augments the formation of declarative video–tone pair memories. Another form of associative learning, classical fear conditioning, models nondeclarative, emotional memory with distinct neuronal mechanisms. Typical fear-conditioning tasks pair a conditioned stimulus (CS) in one modality with an aversive unconditioned stimulus (US) in another. The present study examines the effects of CS–US synchronization in the theta band on fear memory formation in humans. In a fear generalization procedure, we paired one of five visual gratings of varying orientation (CS) with an aversive auditory US. We modulated the luminance of the CS and the volume of the US at a rate of 4 Hz. To manipulate the synchrony between visual and auditory input during fear acquisition, one group (N = 20) received synchronous CS–US pairing, whereas the control group (N = 20) received the CS–US pairs out of phase. Phase synchronization improved CS–US contingency knowledge and facilitated CS discrimination in terms of rated valence and arousal, resulting in narrower generalization across the CS gratings compared with the out-of-phase group. In contrast, synchronization did not amplify conditioned responding in physiological arousal (skin conductance) and visuocortical engagement (steady-state visually evoked potentials) during acquisition, although both measures demonstrated tuning toward the CS+. Together, these data support a causal role of theta-phase synchronization in affective evaluation and contingency report during fear acquisition.

Heart rate pattern and resting heart rate variability mediate individual differences in contextual anxiety and conditioned responses

Past research on backward conditioning in evaluative and fear conditioning yielded inconsistent results in that self-report measures suggest that the conditional stimulus (CS) acquired the valence of the unconditional stimulus (US) in fear conditioning (assimilation effects), but the opposite valence in evaluative conditioning (contrast effects). Conversely, implicit measures of CS valence suggest assimilation effects in evaluative backward conditioning, whereas startle modulation indicates contrast effects in backward fear conditioning. The current study investigated whether US intensity could account for the dissociation on implicit measures between fear and evaluative conditioning. Self-report measures of evaluative learning indicated assimilation effects for forward conditioning, whereas backward contrast effects were observed with intense USs only. Blink startle modulation indicated assimilation effects in forward conditioning and contrast effects in backward conditioning, regardless of US intensity. Experiment 2 included a neutral US in order to assess whether the offset of the positive US elicits an opponent emotional response that mirrors relief (disappointment), which is thought to mediate the reduction in startle seen during backward CSs in fear conditioning. This opponent emotional response was evident as startle magnitude during backward CSs increased linearly with increasing US pleasantness. Omission of the forward CSs led to an assimilation effect in self-report measures. The current results extend our understanding of emotional learning to stimuli encountered after salient emotional events. Startle reflects the emotion prevailing after US offset, relief or disappointment, whereas self-report measures seem more attuned to factors such as US predictability and intensity.

A comparison of neuronal reactions during classical and instrumental conditioning under similar conditions

Neural correlates of fear conditioning are associated with treatment-outcomes to behavioral exposure in spider phobia – Evidence from magnetoencephalography

Emotion–attention interactions in fear conditioning: Moderation by executive load, neuroticism, and awareness

Neonatal basal forebrain cholinergic hypofunction affects ultrasonic vocalizations and fear conditioning responses in preweaning rats

Although previous studies show that the cerebellum is involved in classical fear conditioning, it is not clear which components in the cerebellum control it or how. We addressed this issue using a delayed fear-conditioning paradigm with late-stage zebrafish larvae, with the light extinguishment as the conditioned stimulus (CS) and an electric shock as the unconditioned stimulus (US). The US induced bradycardia in the restrained larvae. After paired-associate conditioning with the CS and US, a substantial population of the larvae displayed CS-evoked bradycardia responses. To investigate the roles of the zebrafish cerebellum in classical fear conditioning, we expressed botulinum toxin or the Ca2+ indicator GCaMP7a in cerebellar neurons. The botulinum-toxin-dependent inhibition of granule-cell transmissions in the corpus cerebelli (CCe, the medial lobe) did not suppress the CS-evoked bradycardia response, but rather prolonged the response. We identified cerebellar neurons with elevated CS-evoked activity after the conditioning. The CS-evoked activity of these neurons was progressively upregulated during the conditioning and was downregulated with repetition of the unpaired CS. Some of these neurons were activated immediately upon the CS presentation, whereas others were activated after a delay. Our findings indicate that granule cells control the recovery from conditioned fear responses in zebrafish.

The role of contingency awareness in simple associative learning experiments with human participants is currently debated. Since prior work suggests that eye movements can index mnemonic processes that occur without awareness, we used eye tracking to better understand the role of awareness in learning aversive Pavlovian conditioning. A complex real-world scene containing four embedded household items was presented to participants while skin conductance, eye movements, and pupil size were recorded. One item embedded in the scene served as the conditional stimulus (CS). One exemplar of that item (e.g. a white pot) was paired with shock 100 percent of the time (CS+) while a second exemplar (e.g. a gray pot) was never paired with shock (CS-). The remaining items were paired with shock on half of the trials. Participants rated their expectation of receiving a shock during each trial, and these expectancy ratings were used to identify when (i.e. on what trial) each participant became aware of the programmed contingencies. Disproportionate viewing of the CS was found both before and after explicit contingency awareness, and patterns of viewing distinguished the CS+ from the CS-. These observations are consistent with “dual process” models of fear conditioning, as they indicate that learning can be expressed in patterns of viewing prior to explicit contingency awareness.

Interval Timing in Aversive Conditioning: Neural Correlates in Amygdala and Related Networks in Rats

Extinction learning refers to a reduction in fear to a conditioned stimulus (CS) that previously signaled a threat, but now occurs without the expected threat. This mechanism is core to exposure-based treatments for anxiety-related disorders. Enhancing the generalization of extinction learning is crucial for improving treatment outcomes, as it helps reduce fear across a range of generalization stimuli (GSs) resembling the original fear-evoking CS. This narrative review identifies and covers three generalization of extinction learning models: 1) generalization of CS extinction learning, examining how extinction learning to the CS generalizes to novel GSs, 2) generalization of GS extinction learning, assessing how extinction learning to a GS generalizes to other novel GSs or the original CS, and 3) generalization of multiple stimuli extinction learning, where extinction learning involves multiple GSs (and sometimes the CS) and its effect on other novel stimuli. While extinction learning to the CS effectively generalizes to other stimuli, extinction learning to a GS or multiple GSs showed limited generalization to other novel GSs or the original CS. Since real-life exposure-based treatment rarely reproduces the CS, extinction learning involving only the GS(s) may better reflect clinical practice; poor generalization of GS(s) extinction learning may constitute another pathway of return of fear. This review also highlights various factors that influence generalization of extinction learning and call for future research to develop strategies for improving these processes, which can help inform exposure-based treatments.