Functional gene polymorphisms in the serotonin system and traumatic life events modulate the neural basis of fear acquisition and extinction

Fear acquisition and extinction are crucial mechanisms in the etiology and maintenance of anxiety disorders. Moreover, they might play a pivotal role in conveying the influence of genetic and environmental factors on the development of a (more or less) stronger proneness for, or resilience against psychopathology. There are only few insights in the neurobiology of genetically and environmentally based individual differences in fear learning and extinction. In this functional magnetic resonance imaging study, 74 healthy subjects were investigated. These were invited according to 5-HTTLPR/rs25531 (S+ vs. L(A)L(A); triallelic classification) and TPH2 (G(-703)T) (T+ vs. T-) genotype. The aim was to investigate the influence of genetic factors and traumatic life events on skin conductance responses (SCRs) and neural responses (amygdala, insula, dorsal anterior cingulate cortex (dACC) and ventromedial prefrontal cortex (vmPFC)) during acquisition and extinction learning in a differential fear conditioning paradigm. Fear acquisition was characterized by stronger late conditioned and unconditioned responses in the right insula in 5-HTTLPR S-allele carriers. During extinction traumatic life events were associated with reduced amygdala activation in S-allele carriers vs. non-carriers. Beyond that, T-allele carriers of the TPH2 (G(-703)T) polymorphism with a higher number of traumatic life events showed enhanced responsiveness in the amygdala during acquisition and in the vmPFC during extinction learning compared with non-carriers. Finally, a combined effect of the two polymorphisms with higher responses in S- and T-allele carriers was found in the dACC during extinction. The results indicate an increased expression of conditioned, but also unconditioned fear responses in the insula in 5-HTTLPR S-allele carriers. A combined effect of the two polymorphisms on dACC activation during extinction might be associated with prolonged fear expression. Gene-by-environment interactions in amygdala and vmPFC activation may reflect a neural endophenotype translating genetic and adverse environmental influences into vulnerability for or resilience against developing affective psychopathology.     

Neural circuitry underlying the regulation of conditioned fear and its relation to extinction

Recent efforts to translate basic research to the treatment of clinical disorders have led to a growing interest in exploring mechanisms for diminishing fear. This research has emphasized two approaches: extinction of conditioned fear, examined across species; and cognitive emotion regulation, unique to humans. Here, we sought to examine the similarities and differences in the neural mechanisms underlying these two paradigms for diminishing fear. Using an emotion regulation strategy, we examine the neural mechanisms of regulating conditioned fear using fMRI and compare the resulting activation pattern with that observed during classic extinction. Our results suggest that the lateral PFC regions engaged by cognitive emotion regulation strategies may influence the amygdala, diminishing fear through similar vmPFC connections that are thought to inhibit the amygdala during extinction. These findings further suggest that humans may have developed complex cognition that can aid in regulating emotional responses while utilizing phylogenetically shared mechanisms of extinction.     

Fear-conditioning mechanisms associated with trait vulnerability to anxiety in humans

Investigations of fear conditioning in rodents and humans have illuminated the neural mechanisms underlying cued and contextual fear. A critical question is how personality dimensions such as trait anxiety act through these mechanisms to confer vulnerability to anxiety disorders, and whether humans' ability to overcome acquired fears depends on regulatory skills not characterized in animal models. In a neuroimaging study of fear conditioning in humans, we found evidence for two independent dimensions of neurocognitive function associated with trait vulnerability to anxiety. The first entailed increased amygdala responsivity to phasic fear cues. The second involved impoverished ventral prefrontal cortical (vPFC) recruitment to downregulate both cued and contextual fear prior to omission (extinction) of the aversive unconditioned stimulus. These two dimensions may contribute to symptomatology differences across anxiety disorders; the amygdala mechanism affecting the development of phobic fear and the frontal mechanism influencing the maintenance of both specific fears and generalized anxiety.     

Amygdala and bed nucleus of the stria terminalis: differential roles in fear and anxiety measured with the acoustic startle reflex.

Neural stimuli associated with traumatic events can readily become conditioned so as to reinstate the memory of the original trauma. These conditioned fear responses can last a lifetime and may be especially resistant to extinction. A large amount of data from many different laboratories indicate that the amygdala plays a crucial role in conditioned fear. The amygdala receives information from all sensory modalities and projects to a variety of hypothalamic and brainstem target areas known to be critically involved in specific signs that are used to define fear and anxiety. Electrical stimulation of the amygdala elicits a pattern of behaviours that mimic natural or conditioned states of fear. Lesions of the amygdala block innate or conditioned fear and local infusion of drugs into the amygdala have anxiolytic effects in several behavioural tests. Excitatory amino acid receptors in the amygdala are critical for the acquisition, expression and extinction of conditioned fear.     

Consolidation of fear extinction requires NMDA receptor-dependent bursting in the ventromedial prefrontal cortex

Extinction of conditioned fear is an active learning process requiring N-methyl-D-aspartate receptors (NMDARs), but the timing, location, and neural mechanisms of NMDAR-mediated processing in extinction are a matter of debate. Here we show that infusion of the NMDAR antagonist CPP into the ventromedial prefrontal cortex (vmPFC) prior to, or immediately after, extinction training impaired 24 hr recall of extinction. These findings indicate that consolidation of extinction requires posttraining activation of NMDARs within the vmPFC. Using multichannel unit recording, we observed that CPP selectively reduced burst firing in vmPFC neurons, suggesting that bursting in vmPFC is necessary for consolidation of extinction. In support of this, we found that the degree of bursting in infralimbic vmPFC neurons shortly after extinction predicted subsequent recall of extinction. We suggest that NMDAR-dependent bursting in the infralimbic vmPFC initiates calcium-dependent molecular cascades that stabilize extinction memory, thereby allowing for successful recall of extinction.     

Directional Theta Coherence in Prefrontal Cortical to Amygdalo-Hippocampal Pathways Signals Fear Extinction

Theta oscillations are considered crucial mechanisms in neuronal communication across brain areas, required for consolidation and retrieval of fear memories. One form of inhibitory learning allowing adaptive control of fear memory is extinction, a deficit of which leads to maladaptive fear expression potentially leading to anxiety disorders. Behavioral responses after extinction training are thought to reflect a balance of recall from extinction memory and initial fear memory traces. Therefore, we hypothesized that the initial fear memory circuits impact behavioral fear after extinction, and more specifically, that the dynamics of theta synchrony in these pathways signal the individual fear response. Simultaneous multi-channel local field and unit recordings were obtained from the infralimbic prefrontal cortex, the hippocampal CA1 and the lateral amygdala in mice. Data revealed that the pattern of theta coherence and directionality within and across regions correlated with individual behavioral responses. Upon conditioned freezing, units were phase-locked to synchronized theta oscillations in these pathways, characterizing states of fear memory retrieval. When the conditioned stimulus evoked no fear during extinction recall, theta interactions were directional with prefrontal cortical spike firing leading hippocampal and amygdalar theta oscillations. These results indicate that the directional dynamics of theta-entrained activity across these areas guide changes in appraisal of threatening stimuli during fear memory and extinction retrieval. Given that exposure therapy involves procedures and pathways similar to those during extinction of conditioned fear, one therapeutical extension might be useful that imposes artificial theta activity to prefrontal cortical-amygdalo-hippocampal pathways that mimics the directionality signaling successful extinction recall.     

Generalization of Human Fear Acquisition and Extinction within a Novel Arbitrary Stimulus Category

Adaptive anxiety relies on a balance between the generalization of fear acquisition and fear extinction. Research on fear (extinction) generalization has focused mostly on perceptual similarity, thereby ignoring the importance of conceptual stimulus relations in humans. The present study used a laboratory procedure to create de novo conceptual categories of arbitrary stimuli and investigated fear and extinction generalization among these stimuli. A matching-to-sample task produced two four-member categories of abstract figures. Next, a member from one category was coupled with an aversive electrical stimulation, while a member from the other category was presented alone. As expected, conditioned fear responses generalized to the other members of the first category (skin conductance and online shock-expectancy). Subsequent extinction of the conditioned member also generalized to the other members. However, extinguishing a non-conditioned member failed to reduce fear of the conditioned member itself. We conclude that fears generalize readily across conceptually related stimuli, but that the degree of extinction generalization depends on the stimulus subjected to extinction.     

Fear Conditioning in an Abdominal Pain Model: Neural Responses during Associative Learning and Extinction in Healthy Subjects

Fear conditioning is relevant for elucidating the pathophysiology of anxiety, but may also be useful in the context of chronic pain syndromes which often overlap with anxiety. Thus far, no fear conditioning studies have employed aversive visceral stimuli from the lower gastrointestinal tract. Therefore, we implemented a fear conditioning paradigm to analyze the conditioned response to rectal pain stimuli using fMRI during associative learning, extinction and reinstatement.In N = 21 healthy humans, visual conditioned stimuli (CS⁺) were paired with painful rectal distensions as unconditioned stimuli (US), while different visual stimuli (CS⁻) were presented without US. During extinction, all CSs were presented without US, whereas during reinstatement, a single, unpaired US was presented. In region-of-interest analyses, conditioned anticipatory neural activation was assessed along with perceived CS-US contingency and CS unpleasantness.Fear conditioning resulted in significant contingency awareness and valence change, i.e., learned unpleasantness of a previously neutral stimulus. This was paralleled by anticipatory activation of the anterior cingulate cortex, the somatosensory cortex and precuneus (all during early acquisition) and the amygdala (late acquisition) in response to the CS⁺. During extinction, anticipatory activation of the dorsolateral prefrontal cortex to the CS⁻ was observed. In the reinstatement phase, a tendency for parahippocampal activation was found.Fear conditioning with rectal pain stimuli is feasible and leads to learned unpleasantness of previously neutral stimuli. Within the brain, conditioned anticipatory activations are seen in core areas of the central fear network including the amygdala and the anterior cingulate cortex. During extinction, conditioned responses quickly disappear, and learning of new predictive cue properties is paralleled by prefrontal activation. A tendency for parahippocampal activation during reinstatement could indicate a reactivation of the old memory trace. Together, these findings contribute to our understanding of aversive visceral learning and memory processes relevant to the pathophysiology of chronic abdominal pain.     

Enhanced Histone Acetylation in the Infralimbic Prefrontal Cortex is Associated with Fear Extinction

The molecular processes that establish fear memory are complex and involve a combination of genetic and epigenetic influences. Dysregulation of these processes can manifest in humans as a range of fear-related anxiety disorders like post-traumatic stress disorders (PTSD). In the present study, immunohistochemistry for acetyl H3, H4, c-fos, CBP (CREB-binding protein) in the infralimbic prefrontal cortex (IL-PFC) and prelimbic prefrontal cortex (PL-PFC) of mPFC (medial prefrontal cortex) and basal amygdala (BA), lateral amygdala (LA), centrolateral amygdala (CeL), centromedial amygdala (CeM) of the amygdala was performed to link region-specific histone acetylation to fear and extinction learning. It was found that the PL-PFC and IL-PFC along with the sub-regions of the amygdala responded differentially to the fear learning and extinction. Following fear learning, c-fos and CBP expression and acetylation of H3 and H4 increased in the BA, LA, CeM, and CeL and the PL-PFC but not in the IL-PFC as compared to the naive control. Similarly, following extinction learning, c-fos and CBP expression increased in BA, LA, CeL, and IL-PFC but not in PL-PFC and CeM as compared to the naive control and conditioned group. However, the acetylation of H3 increased in both IL and PL as opposed to H4 which increased only in the IL-PFC following extinction learning. Overall, region-specific activation in amygdala and PFC following fear and extinction learning as evident by the c-fos activation paralleled the H3/H4 acetylation in these regions. These results suggest that the differential histone acetylation in the PFC and amygdala subnuclei following fear learning and extinction may be associated with the region-specific changes in the neuronal activation pattern resulting in more fear/less fear.     

How engram mediates learning,extinction, and relapse

Fear learning ensures survival through an expression of certain behavior as a conditioned fear response. Fear memory is processed and stored in a fear memory circuit, including the amygdala, hippocampus, and prefrontal cortex. A gradual decrease in conditioned fear response can be induced by fear extinction, which is mediated through the weakening of the original fear memory traces and the newly formed inhibition of those traces. Fear memory can also recover after extinction, which shows flexible control of the fear memory state. Here, we demonstrate how fear engram, which is a physical substrate of fear memory, changes during fear extinction and relapse by reviewing recent studies regarding engram.     

Fear Extinction as a Model for Synaptic Plasticity in Major Depressive Disorder

Background

The neuroplasticity hypothesis of major depressive disorder proposes that a dysfunction of synaptic plasticity represents a basic pathomechanism of the disorder. Animal models of depression indicate enhanced plasticity in a ventral emotional network, comprising the amygdala. Here, we investigated fear extinction learning as a non-invasive probe for amygdala-dependent synaptic plasticity in patients with major depressive disorder and healthy controls.

Methods

Differential fear conditioning was measured in 37 inpatients with severe unipolar depression (International Classification of Diseases, 10^th revision, criteria) and 40 healthy controls. The eye-blink startle response, a subcortical output signal that is modulated by local synaptic plasticity in the amygdala in fear acquisition and extinction learning, was recorded as the primary outcome parameter.

Results

After robust and similar fear acquisition in both groups, patients with major depressive disorder showed significantly enhanced fear extinction learning in comparison to healthy controls, as indicated by startle responses to conditioned stimuli. The strength of extinction learning was positively correlated with the total illness duration.

Conclusions

The finding of enhanced fear extinction learning in major depressive disorder is consistent with the concept that the disorder is characterized by enhanced synaptic plasticity in the amygdala and the ventral emotional network. Clinically, the observation emphasizes the potential of successful extinction learning, the basis of exposure therapy, in anxiety-related disorders despite the frequent comorbidity of major depressive disorder.     

Patterns of coupled theta activity in amygdala-hippocampal-prefrontal cortical circuits during fear extinction

Signals related to fear memory and extinction are processed within brain pathways involving the lateral amygdala (LA) for formation of aversive stimulus associations, the CA1 area of the hippocampus for context-dependent modulation of these associations, and the infralimbic region of the medial prefrontal cortex (mPFC) for extinction processes. While many studies have addressed the contribution of each of these modules individually, little is known about their interactions and how they function as an integrated system. Here we show, by combining multiple site local field potential (LFP) and unit recordings in freely behaving mice in a fear conditioning paradigm, that theta oscillations may provide a means for temporally and functionally connecting these modules. Theta oscillations occurred with high specificity in the CA1-LA-mPFC network. Theta coupling increased between all areas during retrieval of conditioned fear, and declined during extinction learning. During extinction recall, theta coupling partly rebounded in LA-mPFC and CA1-mPFC, and remained at a low level in CA1-LA. Interfering with theta coupling through local electrical microstimulation in CA1-LA affected conditioned fear and extinction recall depending on theta phase. These results support the hypothesis that theta coupling provides a means for inter-areal coordination in conditioned behavioral responsiveness. More specifically, theta oscillations seem to contribute to a population code indicating conditioned stimuli during recall of fear memory before and after extinction.     

Blockade of amygdala metabotropic glutamate receptor subtype 1 impairs fear extinction

The metabotropic glutamate receptor subtype 1 (mGluR1) is thought to be crucial for several forms of memory, but its role in memory extinction has not been determined. Here, we examined a role of mGluR1 in the extinction of conditioned fear using microinjection of an mGluR1 antagonist, CPCCOEt, into the lateral amygdala (LA), a critical structure for fear conditioning and extinction. Intra-LA injection of 3 microg CPCCOEt impaired extinction that was initiated 48 h after the conditioning, but not that initiated 2h after the conditioning, indicating that the effectiveness of CPCCOEt depends upon the length of time since fear conditioning. The CPCCOEt injection failed to alter an mGluR1-like receptor (mGluR5)-dependent acquisition of fear memory, further supporting the specificity of the injected CPCCOEt on mGluR1. Together, our results suggest that amygdala mGluR1 plays a critical role in the extinction of learned fear, but not in the acquisition of fear memory.     

Gastrin-releasing peptide signaling plays a limited and subtle role in amygdala physiology and aversive memory

Links between synaptic plasticity in the lateral amygdala (LA) and Pavlovian fear learning are well established. Neuropeptides including gastrin-releasing peptide (GRP) can modulate LA function. GRP increases inhibition in the LA and mice lacking the GRP receptor (GRPR KO) show more pronounced and persistent fear after single-trial associative learning. Here, we confirmed these initial findings and examined whether they extrapolate to more aspects of amygdala physiology and to other forms of aversive associative learning. GRP application in brain slices from wildtype but not GRPR KO mice increased spontaneous inhibitory activity in LA pyramidal neurons. In amygdala slices from GRPR KO mice, GRP did not increase inhibitory activity. In comparison to wildtype, short- but not long-term plasticity was increased in the cortico-lateral amygdala (LA) pathway of GRPR KO amygdala slices, whereas no changes were detected in the thalamo-LA pathway. In addition, GRPR KO mice showed enhanced fear evoked by single-trial conditioning and reduced spontaneous firing of neurons in the central nucleus of the amygdala (CeA). Altogether, these results are consistent with a potentially important modulatory role of GRP/GRPR signaling in the amygdala. However, administration of GRP or the GRPR antagonist (D-Phe(6), Leu-NHEt(13), des-Met(14))-Bombesin (6-14) did not affect amygdala LTP in brain slices, nor did they affect the expression of conditioned fear following intra-amygdala administration. GRPR KO mice also failed to show differences in fear expression and extinction after multiple-trial fear conditioning, and there were no differences in conditioned taste aversion or gustatory neophobia. Collectively, our data indicate that GRP/GRPR signaling modulates amygdala physiology in a paradigm-specific fashion that likely is insufficient to generate therapeutic effects across amygdala-dependent disorders.     

Neuropeptide S-mediated control of fear expression and extinction: role of intercalated GABAergic neurons in the amygdala

A deficient extinction of memory is particularly important in the regime of fear, where it limits the beneficial outcomes of treatments of anxiety disorders. Fear extinction is thought to involve inhibitory influences of the prefrontal cortex on the amygdala, although the detailed synaptic mechanisms remain unknown. Here, we report that neuropeptide S (NPS), a recently discovered transmitter of ascending brainstem neurons, evokes anxiolytic effects and facilitates extinction of conditioned fear responses when administered into the amygdala in mice. An NPS receptor antagonist exerts functionally opposing responses, indicating that endogenous NPS is involved in anxiety behavior and extinction. Cellularly, NPS increases glutamatergic transmission to intercalated GABAergic neurons in the amygdala via presynaptic NPS receptors on connected principal neurons. These results identify mechanisms of NPS in the brain, a key role of intercalated neurons in the amygdala for fear extinction, and a potential pharmacological avenue for treating anxiety disorders.     

Effects of Methamphetamine Exposure on Fear Learning and Memory in Adult and Adolescent Rats

Methamphetamine (meth) use is often comorbid with anxiety disorders, with both conditions predominant during adolescence. Conditioned fear extinction is the most widely used model to study the fear learning and regulation that are relevant for anxiety disorders. The present study investigates how meth binge injections or meth self-administration affect subsequent fear conditioning, extinction and retrieval in adult and adolescent rats. In experiment 1, postnatal day 35 (P35—adolescent) and P70 (adult) rats were intraperitoneally injected with increasing doses of meth across 9 days. At P50 or P85, they underwent fear conditioning followed by extinction and test. In experiments 2a–c, P35 or P70 rats self-administered meth for 11 days then received fear conditioning at P50 or P85, followed by extinction and test. We observed that meth binge exposure caused a significant disruption of extinction retrieval in adult but not adolescent rats. Interestingly, meth self-administration in adolescence or adulthood disrupted acquisition of conditioned freezing in adulthood. Meth self-administration in adolescence did not affect conditioned freezing in adolescence. These results suggest that intraperitoneal injections of high doses of meth and meth self-administration have dissociated effects on fear conditioning and extinction during adulthood, while adolescent fear conditioning and extinction are unaffected.

     

Neuronal signalling of fear memory

The learning and remembering of fearful events depends on the integrity of the amygdala, but how are fear memories represented in the activity of amygdala neurons? Here, we review recent electrophysiological studies indicating that neurons in the lateral amygdala encode aversive memories during the acquisition and extinction of Pavlovian fear conditioning. Studies that combine unit recording with brain lesions and pharmacological inactivation provide evidence that the lateral amygdala is a crucial locus of fear memory. Extinction of fear memory reduces associative plasticity in the lateral amygdala and involves the hippocampus and prefrontal cortex. Understanding the signalling of aversive memory by amygdala neurons opens new avenues for research into the neural systems that support fear behaviour.     

Context-dependent encoding of fear and extinction memories in a large-scale network model of the basal amygdala

The basal nucleus of the amygdala (BA) is involved in the formation of context-dependent conditioned fear and extinction memories. To understand the underlying neural mechanisms we developed a large-scale neuron network model of the BA, composed of excitatory and inhibitory leaky-integrate-and-fire neurons. Excitatory BA neurons received conditioned stimulus (CS)-related input from the adjacent lateral nucleus (LA) and contextual input from the hippocampus or medial prefrontal cortex (mPFC). We implemented a plasticity mechanism according to which CS and contextual synapses were potentiated if CS and contextual inputs temporally coincided on the afferents of the excitatory neurons. Our simulations revealed a differential recruitment of two distinct subpopulations of BA neurons during conditioning and extinction, mimicking the activation of experimentally observed cell populations. We propose that these two subgroups encode contextual specificity of fear and extinction memories, respectively. Mutual competition between them, mediated by feedback inhibition and driven by contextual inputs, regulates the activity in the central amygdala (CEA) thereby controlling amygdala output and fear behavior. The model makes multiple testable predictions that may advance our understanding of fear and extinction memories.     

Behavioral and neural analysis of extinction

The neural mechanisms by which fear is inhibited are poorly understood at the present time. Behaviorally, a conditioned fear response may be reduced in intensity through a number of means. Among the simplest of these is extinction, a form of learning characterized by a decrease in the amplitude and frequency of a conditioned response when the conditioned stimulus that elicits it is repeatedly nonreinforced. Because clinical interventions for patients suffering from fear dysregulation seek to inhibit abnormal, presumably learned fear responses, an understanding of fear extinction is likely to inform and increase the efficacy of these forms of treatment. This review considers the behavioral, cellular, and molecular literatures on extinction and presents the most recent advances in our understanding while identifying issues that require considerable further research.     

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.