Positron emission tomographic imaging of neural correlates of a fear acquisition and extinction paradigm in women with childhood sexual-abuse-related post-traumatic stress disorder

BACKGROUND: In the conditioned fear paradigm, repeated pairing of an aversive unconditioned stimulus (US) (e.g. electric shock) with a neutral conditioned stimulus (CS) (e.g. bright light) results in a conditioned fear response to the light alone. Animal studies have shown that the amygdala plays a critical role in acquisition of conditioned fear responses, while the medial prefrontal cortex (including anterior cingulate), through inhibition of amygdala responsiveness, has been hypothesized to play a role in extinction of fear responses. No studies have examined neural correlates of fear conditioning and extinction in patients with post-traumatic stress disorder (PTSD). METHOD: Women with early childhood sexual-abuse-related PTSD (n = 8) and women without abuse or PTSD (n = 11) underwent measurement of psychophysiological (skin conductance) responding as well as positron emission tomographic (PET) measurement of cerebral blood flow during habituation, acquisition and extinction conditions. During habituation subjects were repeatedly exposed to a blue square on a screen. During acquisition, exposure to the blue square (CS) was paired with an electric shock to the forearm (US). With extinction, subjects were again exposed to the blue squares without shock. On a different day subjects went through the same procedure with electric shocks administered randomly in the absence of the blue square. RESULTS: Skin conductance responding to the CS was consistent with the development of conditioned responses with this paradigm. PTSD patients had increased left amygdala activation with fear acquisition, and decreased anterior cingulate function during extinction, relative to controls. CONCLUSIONS: These findings implicate amygdala and anterior cingulate in the acquisition and extinction of fear responses, respectively, in PTSD.     

The lateral amygdala processes the value of conditioned and unconditioned aversive stimuli

The amygdala is critical for acquiring and expressing conditioned fear responses elicited by sensory stimuli that predict future punishment, but there is conflicting evidence about whether the amygdala is necessary for perceiving the aversive qualities of painful or noxious stimuli that inflict primary punishment. To investigate this question, rats were fear conditioned by pairing a sequence of auditory pips (the conditioned stimulus, or CS) with a brief train of shocks to one eyelid (the unconditioned stimulus, or US). Conditioned responding to the CS was assessed by measuring freezing responses during a test session conducted 24 h after training, and unconditioned responding to the US was assessed by measuring head movements evoked by the eyelid shocks during training. We found that pre-training electrolytic lesions of the amygdala's lateral (LA) nucleus blocked acquisition of conditioned freezing to the CS, and also significantly attenuated unconditioned head movements evoked by the US. Similarly, bilateral inactivation of the amygdala with the GABA-A agonist muscimol impaired acquisition of CS-evoked freezing, and also attenuated US-evoked responses during training. However, when amygdala synaptic plasticity was blocked by infusion of the NR2B receptor antagonist ifenprodil, acquisition of conditioned freezing was impaired but shock reactivity was unaffected. These findings indicate that neural activity within the amygdala is important for both predicting and perceiving the aversive qualities of noxious stimuli, and that synaptic plasticity within LA is the mechanism by which the CS becomes associated with the US during fear conditioning.     

Conditioned and extinguished fear modulate functional corticocardiac coupling in humans

Although the conditioned cardiac fear response is an important index of psychophysiological fear processing, underlying neural mechanisms remain unclear. N = 22 participants underwent differential fear conditioning and extinction with face pictures as conditioned stimuli (CS) and loud noise bursts as aversive unconditioned stimulus (US) on Day 1 and a recall test 1 day later. We assessed ERPs, evoked heart period (HP), and time‐lagged within‐subject correlations of single‐trial EEG amplitude and HP as index for corticocardiac coupling in response to the CS. Fear‐conditioned stimuli (CS+) triggered cardiac deceleration during fear acquisition and recall. Meanwhile, only during Day 1 acquisition, CS+ evoked larger late positivities in the ERP than CS?. Most importantly, during Day 2 recall, stimulus‐evoked single‐trial EEG responses in the time window between 250 and 500 ms predicted the magnitude of cardiac fear responses 2 to 5 s later. This marker of corticocardiac coupling selectively emerged in response to not previously extinguished CS+ but was absent in response to CS? or previously extinguished CS+. The present results provide first evidence that fear conditioning and extinction modulate functional corticocardiac coupling in humans. Underlying mechanisms may involve subcortical structures enhancing corticocardiac transmission to facilitate processing of consolidated conditioned fear.     

Learning to fear suffocation: a new paradigm for interoceptive fear conditioning

The present study aimed to establish a new interoceptive fear conditioning paradigm. The conditioned stimulus (CS) was a flow resistor that slightly obstructs breathing; the unconditional stimulus (US) was a breathing occlusion. The paired group (N = 21) received 6 acquisition trials with paired CS–US presentations. The unpaired group (N = 19) received 6 trials of unpaired CS–US presentations. In the extinction phase, both groups were administered 6 CS‐only trials. Measurements included startle eyeblink response, electrodermal responses, and self‐reported US expectancy. In the paired group, startle blink responses were larger during CS compared to intertrial interval during acquisition and extinction. Electrodermal and US expectancies were larger for the paired than for the unpaired group during acquisition, but not during extinction. The present paradigm successfully established interoceptive fear conditioning with panic‐relevant stimuli.     

The dopaminergic modulation of fear: quinpirole impairs the recall of emotional memories in rats

Past studies examining the contributions of dopamine to fear have produced inconsistent results. The present experiments reevaluated this issue. It was found that systemic pretreatment with the D2 agonist quinpirole before pairing 2 conditioned stimuli (CSs; CS2-CS1) dose dependently blocked the acquisition of second-order fear conditioning. Quinpirole's actions were not due to nonspecific impairments in the ability to perceive the CSs, or form and store an association, because the identical drug pretreatment before pairing the same 2 CSs had no effect on the acquisition of sensory preconditioning. In a separate study, rats were given fear conditioning while untreated and then received extinction sessions while under the influence of quinpirole or its vehicle. Quinpirole pretreatment blocked extinction. Findings suggest that quinpirole decreased fear by blocking the retrieval of a learned association between a CS and unconditioned stimulus (US), rather than by devaluing the US, which would have resulted from summation of quinpirole's appetitive properties with the aversive properties of fear.     

Arousal-related associative response characteristics of dorsal lateral geniculate nucleus neurons during acoustic Pavlovian fear conditioning

This research determined whether fear-conditioned, acoustic stimuli induce thalamic arousal reflected in associative responses in dorsal lateral geniculate nucleus (dLGN) neurons. Rabbits received a Pavlovian discriminative fear conditioning procedure in which one tone conditioned stimulus (CS +) was always paired with an aversive unconditioned stimulus (US) and another tone (CS-) was never paired with the US. Responses of single dLGN neurons to random CS+ and CS- presentations were then recorded. Nine of 15 recorded neurons demonstrated significantly greater firing during the CS+ versus the CS-. Their spontaneous activity demonstrated tonic firing during increased neocortical arousal and burst firing during decreased neocortical arousal. The results demonstrate that dLGN neurons show associative responses to fear-conditioned, acoustic stimuli and present a model for investigating the neural circuits by which such stimuli affect sensory processing at the thalamic level.     

Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning.

The contribution of the amygdala and hippocampus to the acquisition of conditioned fear responses to a cue (a tone paired with footshock) and to context (background stimuli continuously present in the apparatus in which tone-shock pairings occurred) was examined in rats. In unoperated controls, responses to the cue conditioned faster and were more resistant to extinction than were responses to contextual stimuli. Lesions of the amygdala interfered with the conditioning of fear responses to both the cue and the context, whereas lesions of the hippocampus interfered with conditioning to the context but not to the cue. The amygdala is thus involved in the conditioning of fear responses to simple, modality-specific conditioned stimuli as well as to complex, polymodal stimuli, whereas the hippocampus is only involved in fear conditioning situations involving complex, polymodal events. These findings suggest an associative role for the amygdala and a sensory relay role for the hippocampus in fear conditioning.     

Effect of naloxone on conditioned suppression in rats

Painful stimuli are known to engage an endorphin analgesic system that can be reversed by the opiate antagonist, naloxone. Naloxone, then, should increase the effectiveness of aversive unconditioned stimuli (USs) in Pavlovian fear conditioning. Consistent with this hypothesis, naloxone administered during the acquisition of conditioned suppression in rats enhanced posttrial suppression and preconditioned stimulus (pre-CS; context-controlled) suppression. Furthermore, it enhanced CS-elicited suppression during extinction when administered during acquisition but not when administered only during extinction. Thus naloxone does not enhance an already existing fear nor enhance the memory of previous conditioning; instead, it enhances the conditioning of fear presumably by making the aversive US more painful.     

Fear extinction in rats: implications for human brain imaging and anxiety disorders

Fear extinction is the decrease in conditioned fear responses that normally occurs when a conditioned stimulus (CS) is repeatedly presented in the absence of the aversive unconditioned stimulus (US). Extinction does not erase the initial CS-US association, but is thought to form a new memory. After extinction training, extinction memory competes with conditioning memory for control of fear expression. Deficits in fear extinction are thought to contribute to post-traumatic stress disorder (PTSD). Herein, we review studies performed in rats showing that the medial prefrontal cortex plays a critical role in the retention and expression of extinction memory. We also review human studies indicating that prefrontal areas homologous to those critical for extinction in rats are structurally and functionally deficient in patients with PTSD. We then discuss how findings from rat studies may allow us to: (1) develop new fear extinction paradigms in humans, (2) make specific predictions as to the location of extinction-related areas in humans, and (3) improve current extinction-based behavioral therapies for anxiety disorders.     

Generalization of fear in post-traumatic stress disorder

Overgeneralization (i.e., the transfer of fear to stimuli not related to an aversive event) is part of alterations in associative fear learning in mental disorders. In the present experimental study, we investigated whether this holds true for post-traumatic stress disorder (PTSD) related to childhood abuse. We expected that fear generalization under experimental conditions reflects generalization of aversive stimuli to different social domains in real life. Sixty-four women with PTSD after childhood abuse and 30 healthy participants (HC) underwent a differential fear conditioning and generalization paradigm. Online risk ratings, reaction time, and fear-potentiated startle served as dependent variables. Based on the subjectively assessed generalization of triggered intrusions across different domains of life, PTSD participants were split into two groups reporting low (low-GEN) and high (high-GEN) generalization. PTSD patients reported a higher expectation of an aversive event. During fear conditioning, they assessed the risk of danger related to a safety cue slower and showed a blunted fear-potentiated startle toward the danger cue. During generalization testing, reaction time increased in the high-GEN patients and decreased in the HC group with increasing similarity of a stimulus with the conditioned safety cue. Alterations of fear learning in PTSD suggest impaired defensive responses in case of a high threat probability. Moreover, our findings bridge the gap between the generalization of aversive cues during everyday life and laboratory-based experimental parameters: impairments in the processing of cues signaling safety generalize particularly in those patients who report a spreading of PTSD symptoms across different domains of everyday life.     

Mesolimbic dopaminergic pathways in fear conditioning

One of the most common paradigms used to study the biological basis of emotion, as well as of learning and memory, is Pavlovian fear conditioning. In the acquisition phase of a fear conditioning experiment, an emotionally neutral conditioned stimulus (CS)--which can either be a discrete stimulus, such as a tone, or a contextual stimulus, such as a specific environment--is paired with an aversive unconditioned stimulus (US), for example a foot shock. As a result, the CS elicits conditioned fear responses when subsequently presented alone during the expression phase of the experiment. While considerable work has been done in relating specific circuits of the brain to fear conditioning, less is known about its regulation by neuromodulators; the understanding of which would be of therapeutic relevance for fear related diseases such as phobia, panic attacks, post traumatic stress disorder, obsessive compulsive disorder, or generalized anxiety disorder. Dopamine is one of the neuromodulators most potently acting on the mechanisms underlying states of fear and anxiety. Recently, a growing body of evidence has suggested that dopaminergic mechanisms are significant for different aspects of affective memory, namely its formation, expression, retrieval, and extinction. The aim of this review is to clarify the complex actions of dopamine in fear conditioning with respect to the wide-spread distribution of dopaminergic innervation over structures constituting the fear related circuitry. A particular effort is made to understand how dopamine in the amygdala, medial prefrontal cortex and nucleus accumbens--target structures of the mesolimbic dopamine system originating from the ventral tegmental area--could relate to different aspects of fear conditioning.     

Aversive Pavlovian conditioning in psychopaths: peripheral and central correlates

Differential aversive Pavlovian conditioning with a foul odor as unconditioned stimulus (US) and neutral faces as conditioned stimuli (CS) was compared between 9 noncriminal psychopaths as defined by the Hare Psychopathy Checklist Revised and 12 healthy controls. Event-related potentials (ERP), heart rate, skin conductance response, corrugator EMG, and startle response potentiation as well as valence, arousal, and contingency of the CS were assessed. Whereas the healthy controls (HC) showed significant CS +/CS- differentiation, the psychopaths (PP) failed to exhibit a conditioned response although unconditioned responses were comparable between the groups. N100, P200, and P300 to the CSs revealed that psychopaths were not deficient in information processing and showed even better anticipatory responding than the HC group indicated by the terminal contingent negative variation (tCNV), that lacked, however, CS+ and CS- differentiation. These data indicate a deficit in association formation in psychopaths that may be related to deficient interaction of limbic-subcortical and cortical structures.     

Brain circuits involved in emotional learning in antisocial behavior and social phobia in humans

While psychopaths (PP) lack anticipatory fear, social phobics (SP) are characterized by excessive fear. Criminal PP, SP and healthy controls (HC) participated in differential aversive delay conditioning with neutral faces as conditioned (CS) and painful pressure as unconditioned stimuli. Functional magnetic resonance imaging revealed differential activation in the limbic-prefrontal circuit (orbitofrontal cortex, insula, anterior cingulate, amygdala) in the HC. By contrast, the PP displayed brief amygdala, but no further brain activation. The SP showed increased activity to the faces in the amygdala and orbitofrontal cortex already during habituation. Thus, a hypoactive frontolimbic circuit may represent the neural correlate of psychopathic behavior, whereas an overactive frontolimbic system may underly social fear.     

Modeling startle eyeblink electromyogram to assess fear learning

Pavlovian fear conditioning is widely used as a laboratory model of associative learning in human and nonhuman species. In this model, an organism is trained to predict an aversive unconditioned stimulus from initially neutral events (conditioned stimuli, CS). In humans, fear memory is typically measured via conditioned autonomic responses or fear‐potentiated startle. For the latter, various analysis approaches have been developed, but a systematic comparison of competing methodologies is lacking. Here, we investigate the suitability of a model‐based approach to startle eyeblink analysis for assessment of fear memory, and compare this to extant analysis strategies. First, we build a psychophysiological model (PsPM) on a generic startle response. Then, we optimize and validate this PsPM on three independent fear‐conditioning data sets. We demonstrate that our model can robustly distinguish aversive (CS+) from nonaversive stimuli (CS‐, i.e., has high predictive validity). Importantly, our model‐based approach captures fear‐potentiated startle during fear retention as well as fear acquisition. Our results establish a PsPM‐based approach to assessment of fear‐potentiated startle, and qualify previous peak‐scoring methods. Our proposed model represents a generic startle response and can potentially be used beyond fear conditioning, for example, to quantify affective startle modulation or prepulse inhibition of the acoustic startle response.     

Activation of the ventral striatum during aversive contextual conditioning in humans

The goal of this study was to investigate the function of the ventral striatum and brain regions involved in anxiety and learning during aversive contextual conditioning. Functional magnetic resonance imaging was used to assess the hemodynamic brain response of 118 healthy volunteers during a differential fear conditioning paradigm. Concurrently obtained skin conductance responses and self-reports indicated successful context conditioning. Increased hemodynamic responses in the ventral striatum during presentation of the conditioned visual stimulus that predicted the aversive event (CS+) compared to a second stimulus never paired with the aversive event (CS-) were observed in the late acquisition phase. Additionally, we found significant brain responses in the amygdala, hippocampus, insula and medial prefrontal cortex. Our data suggest the involvement of the ventral striatum during contextual fear conditioning, and underline its role in the processing of salient stimuli in general, not only during reward processing.     

The role of prefrontal cortex in predictive fear learning

Pavlovian fear conditioning depends on prediction error, or the discrepancy between actual and expected outcomes. We used immunohistochemistry, neuronal tract tracing, and reversible inactivation to study the role of prefrontal cortex and thalamocortical pathways in predictive fear learning. Unexpected, but not expected, conditioned stimulus (CS)-unconditioned stimulus (US) presentations caused increased c-Fos expression in the prefrontal cortex (PFC), midline thalamus, lateral amygdala, as well as retrograde labeled midline thalamic afferents to PFC. Reversible inactivation of dorsomedial PFC, but not infralimbic PFC, prevented the associative blocking of fear learning. These results suggest a role for dorsomedial PFC (dmPFC), and a thalamic → dmPFC pathway, in signaling whether or not aversive events are expected or unexpected and so whether they are to be learned about.     

A pupil size response model to assess fear learning

During fear conditioning, pupil size responses dissociate between conditioned stimuli that are contingently paired (CS+) with an aversive unconditioned stimulus, and those that are unpaired (CS‐). Current approaches to assess fear learning from pupil responses rely on ad hoc specifications. Here, we sought to develop a psychophysiological model (PsPM) in which pupil responses are characterized by response functions within the framework of a linear time‐invariant system. This PsPM can be written as a general linear model, which is inverted to yield amplitude estimates of the eliciting process in the central nervous system. We first characterized fear‐conditioned pupil size responses based on an experiment with auditory CS. PsPM‐based parameter estimates distinguished CS+/CS‐ better than, or on par with, two commonly used methods (peak scoring, area under the curve). We validated this PsPM in four independent experiments with auditory, visual, and somatosensory CS, as well as short (3.5 s) and medium (6 s) CS/US intervals. Overall, the new PsPM provided equal or decisively better differentiation of CS+/CS‐ than the two alternative methods and was never decisively worse. We further compared pupil responses with concurrently measured skin conductance and heart period responses. Finally, we used our previously developed luminance‐related pupil responses to infer the timing of the likely neural input into the pupillary system. Overall, we establish a new PsPM to assess fear conditioning based on pupil responses. The model has a potential to provide higher statistical sensitivity, can be applied to other conditioning paradigms in humans, and may be easily extended to nonhuman mammals.     

Brain morphology correlates of interindividual differences in conditioned fear acquisition and extinction learning

The neural circuits underlying fear learning have been intensively investigated in pavlovian fear conditioning paradigms across species. These studies established a predominant role for the amygdala in fear acquisition, while the ventromedial prefrontal cortex (vmPFC) has been shown to be important in the extinction of conditioned fear. However, studies on morphological correlates of fear learning could not consistently confirm an association with these structures. The objective of the present study was to investigate if interindividual differences in morphology of the amygdala and the vmPFC are related to differences in fear acquisition and extinction learning in humans. We performed structural magnetic resonance imaging in 68 healthy participants who underwent a differential cued fear conditioning paradigm. Volumes of subcortical structures as well as cortical thickness were computed by the semi-automated segmentation software Freesurfer. Stronger acquisition of fear as indexed by skin conductance responses was associated with larger right amygdala volume, while the degree of extinction learning was positively correlated with cortical thickness of the right vmPFC. Both findings could be conceptually replicated in an independent sample of 53 subjects. The data complement our understanding of the role of human brain morphology in the mechanisms of the acquisition and extinction of conditioned fear.     

Measuring the conditioned response: A comparison of pupillometry,skin conductance,and startle electromyography

In human fear conditioning studies, different physiological readouts can be used to track conditioned responding during fear learning. Commonly employed readouts such as skin conductance responses (SCR) or startle responses have in recent years been complemented by pupillary readouts, but to date it is unknown how pupillary readouts relate to other measures of the conditioned response. To examine differences and communalities among pupil responses, SCR, and startle responses, we simultaneously recorded pupil diameter, skin conductance, and startle electromyography in 47 healthy subjects during fear acquisition, extinction, and a recall test on 2 consecutive days. The different measures correlated only weakly, displaying most prominent differences in their response patterns during fear acquisition. Whereas SCR and startle responses habituated, pupillary measures did not. Instead, they increased in response to fear conditioned stimuli and most closely followed ratings of unconditioned stimulus (US) expectancy. Moreover, we observed that startle‐induced pupil responses showed stimulus discrimination during fear acquisition, suggesting a fear potentiation of the auditory pupil reflex. We conclude that different physiological outcome measures of the conditioned response inform about different cognitive‐affective processes during fear learning, with pupil responses being least affected by physiological habituation and most closely following US expectancy.     

Resting heart rate variability is associated with inhibition of conditioned fear

Startle blink as well as skin conductance responses (SCR) are widely used indices of learning processes associated with fear conditioning and extinction. During safety learning, the amygdala is under top‐down inhibitory control by the prefrontal cortex (PFC). The capacity of the PFC to exert inhibitory control over subcortical brain structures may be indexed by resting state vagally mediated heart rate variability (HRV). The present study investigated the association of resting HRV with startle blink and SCR during conditioned fear inhibition and extinction. Participants first learned to discriminate a threat cue (A) signaling an aversive unconditioned stimulus from a safety signal (B), which were each presented together with a third stimulus X (AX+/BX?). Then, both the threat and safety signal were presented together (AB) to test whether the presence of the learned safety signal inhibits the fear response to the danger signal. Finally, AX was presented without reinforcement (AX?) to investigate fear extinction. Higher HRV was associated with pronounced fear inhibition and fear extinction. Resting HRV levels were associated with fear extinction as indexed by startle blink potentiation but not SCR, which presumably reflect more cognitive aspects of learning. Resting HRV may reflect the capacity of the prefrontal cortex to inhibit subcortical fear responses in the presence of safety or when former threat cues are presented in the absence of threat.