From extinction learning to anxiety treatment: mind the gap

Laboratory models of extinction learning in animals and humans have the potential to illuminate methods for improving clinical treatment of fear-based clinical disorders. However, such translational research often neglects important differences between threat responses in animals and fear learning in humans, particularly as it relates to the treatment of clinical disorders. Specifically, the conscious experience of fear and anxiety, along with the capacity to deliberately engage top-down cognitive processes to modulate that experience, involves distinct brain circuitry and is measured and manipulated using different methods than typically used in laboratory research. This paper will identify how translational research that investigates methods of enhancing extinction learning can more effectively model such elements of human fear learning, and how doing so will enhance the relevance of this research to the treatment of fear-based psychological disorders.Published versio

Functional and neural mechanisms of human fear conditioning: studies in healthy and brain-damaged individuals

Fear conditioning represents the learning process by which a stimulus, after repeated pairing with an aversive event, comes to evoke fear and becomes intrinsically aversive. This learning is essential to organisms throughout the animal kingdom and represents one the most successful laboratory paradigm to reveal the psychological processes that govern the expression of emotional memory and explore its neurobiological underpinnings. Although a large amount of research has been conducted on the behavioural or neural correlates of fear conditioning, some key questions remain unanswered. Accordingly, this thesis aims to respond to some unsolved theoretic and methodological issues, thus furthering our understanding of the neurofunctional basis of human fear conditioning both in healthy and brain-damaged individuals. Specifically, in this thesis, behavioural, psychophysiological, lesion and non-invasive brain stimulation studies were reported. Study 1 examined the influence of normal aging on context-dependent recall of extinction of fear conditioned stimulus. Study 2 aimed to determine the causal role of the ventromedial PFC (vmPFC) in the acquisition of fear conditioning by systematically test the effect of bilateral vmPFC brain-lesion. Study 3 aimed to interfere with the reconsolidation process of fear memory by the means of non-invasive brain stimulation (i.e. TMS) disrupting PFC neural activity. Finally, Study 4 aimed to investigate whether the parasympathetic – vagal – modulation of heart rate might reflect the anticipation of fearful, as compared to neutral, events during classical fear conditioning paradigm. Evidence reported in this PhD thesis might therefore provide key insights and deeper understanding of critical issues concerning the neurofunctional mechanisms underlying the acquisition, the extinction and the reconsolidation of fear memories in humans

Antidepressant-Like Effects of Ketamine on Fear Conditioning and Extinction

The experience of chronic stress plays an important role in the pathogenesis of major depressive disorder. Prolonged stress induces a state of chronically elevated glucocorticoid exposure in the brain, which is neurotoxic and can lead to the dysfunction of glutamatergic signaling. Since memory is highly dependent upon glutamatergic neurotransmission, patients with depression commonly display alterations in memory processing that bias the recollection of past events towards negative emotional information. Negative cognitive biases are believed to support the development and maintenance of depression, emphasizing the need for antidepressant treatments that can effectively combat these insidious symptoms. Memory for negative emotional events can be studied in animals using fear conditioning and extinction paradigms. Fear conditioning trains an animal to associate a neutral cue with an emotionally aversive experience, while extinction learning challenges this association by forming a new memory that identifies the cue as harmless. The aim of this dissertation was to investigate the antidepressant-like effects of ketamine on auditory fear conditioning and extinction in both healthy rats and those with chronic glucocorticoid exposure. The first experiment sought to dissociate the effects of ketamine on distinct stages of auditory fear conditioning and extinction by administering a subanesthetic dose of ketamine at one of three unique time points: before fear conditioning training, immediately after fear conditioning training, or before fear extinction training. Post-conditioning and pre- extinction ketamine attenuated the long-term expression of cue-elicited freezing, suggesting that the consolidation and recall of conditioned fear were impaired, respectively. Pre-conditioning ketamine did not disrupt the acquisition of fear conditioning, and none of the treatments examined affected the long-term expression of fear extinction. The second experiment aimed to build upon these findings by examining the effect of pre-extinction ketamine on conditioned fear and extinction behavior in a repeated exogenous corticosterone (CORT) animal model of depression. Repeated CORT treatment provoked a spontaneous recovery of conditioned freezing between extinction sessions and induced a reinstatement of freezing following a sub- conditioning retraining procedure. Ketamine prevented CORT-induced failures in long-term extinction expression, and also greatly reduced freezing during early phase extinction training. Collectively, the findings of this dissertation help establish ketamine as a powerful modulator of negatively-valenced memory and emotionally-driven behavior, and contribute to our understanding of its antidepressant-like qualities

Neural Circuits Underlying Context-Dependent Fear Memory Retrieval

Exposure therapy for stress- and anxiety-related disorders is very effective, but fear return after treatment is not uncommon. During extinction, repeated exposure to a conditioned stimulus (CS) gradually decreases the probability and magnitude of the conditioned fear response (CR). However, extinction does not eliminate the fear memory; rather, it generates a new extinction memory that competes with the fear memory for control of behavior. Importantly, the extinction memory is highly contextdependent insofar as it is only expressed in the extinction context. That is, if the CS is encountered outside of the extinction context, the conditioned fear response returns or ‘‘renews’’. The renewal of extinguished fear is a considerable challenge for maintaining long-lasting fear suppression after exposure therapy. The hippocampus, the medial prefrontal cortex (mPFC) and the amygdala are thought to play essential roles for context-dependent memory retrieval after extinction, but the circuit mechanism is not clear. To explore the neural circuits underlying contextual regulation of fear memory retrieval, we first used functional tracing approach to examine the activity of prelimbic prefrontal cortex (PL)- and basal amygdala (BA)-projecting neurons in the ventral hippocampus (vHPC) during extinction retrieval and fear renewal. We then used circuitspecific chemogenetic approach to examine the role of infralimbic prefrontal cortex (IL)- projecting vHPC neurons in contextual retrieval. Finally, we used pharmacological and chemogenetic approaches to test the role of the nucleus reuniens (RE) in contextdependent fear memory retrieval. The results showed that vHPC inputs to both the PL and BA are activated during fear renewal, with PL- and BA-dual projecting neurons showing the greatest level of activation. Moreover, we showed that IL-projecting vHPC neurons are required for fear renewal by inducing feedforward inhibition within the IL. Lastly, we demonstrated that the RE is required for extinction retrieval, and the prefrontal modulation on the RE activity is essential in this process. Together, these results provided circuit mechanisms underlying context-dependent fear memory retrieval

Changes in Resting-State Functional Connectivity Following Delay and Trace Fear Conditioning Acquisition and Extinction

Consolidation is the process of stabilizing a recently acquired memory into a more permanent or durable form. Several studies with laboratory animals have uncovered valuable information about the process of consolidation, but less is known about the process of consolidation in healthy humans. The current study examined the consolidation of emotional memories in different brain circuits in healthy humans using resting-state fMRI. We used the acquisition and extinction of two variations of Pavlovian fear conditioning, delay and trace, which rely on slightly different circuits to examine changes in functional connectivity related to a general fear learning process and also to examine how these changes may differ in these circuits. We found that the acquisition of delay and trace fear conditioning involves similar circuitry including the amygdala, but that trace conditioning involves the addition of a few more brain regions to the general circuit including the hippocampus. Twenty-four hours following acquisition there was an increase in functional connectivity between the amygdala and several other brain areas including the hippocampus and medial prefrontal cortex for both the delay and trace groups suggesting that these changes reflect the consolidation of a general fear memory. We also observed changes in connectivity that were specific to the trace group in brain regions thought to be specifically involved in trace conditioning including the medial prefrontal cortex and the retrosplenial cortex. By seven days after acquisition most of the changes in connectivity had returned to baseline. Extinction data revealed that the ventromedial prefrontal cortex was involved in forming this inhibitory memory and that connectivity between the amygdala and a region of ventromedial prefrontal cortex increased for the trace group following extinction. These results suggest that consolidation can be measured in healthy humans using resting-state fMRI and that these processes occur in the same circuits that are responsible during training

Contextual Modulation of Associative Learning and the Role of Resting State Brain Activity in Posttraumatic Stress Disorder

In the present dissertation we addressed neuronal changes in PTSD using an activationbasedand a resting state-based approach with a special focus on brain areas involved in abnormal activation in PTSD such as amygdala, hippocampus, ventromedial prefrontalcortex (vmPFC), dorsal anterior cingulate cortex (dACC) and insula. Our attention was directed to the mechanisms mediating increased return of fear and the association of PTSD symptoms with aberrant brain activity as well as aberrant resting state connectivity. In both studies we compared PTSD patients with trauma-exposed but unaffected controls (non-PTSD) and trauma-naïve healthy controls (HC). In the first study, subjects underwent an ABC fear conditioning and extinction procedure, where two CSs were presented in front of virtual reality scenes. One of them (CS+) was paired with a slightly painful electrical stimulation (US) during acquisition, whereas the other one was never paired with the US (CS-). During extinction, there were no CS-US pairings. After acquisition (context A) and extinction (context B), the participants were brought to a novel context C and again confronted with the CSs. Selfreports, skin conductance responses (SCR) and functional magnetic resonance imaging (fMRI) were measured simultaneously. We found elevated return of fear in the PTSD patients indicated by larger differential SCR compared to non-PTSD and HC and larger differential amygdala and hippocampus activity compared to HC. Increased amygdala activation was positively correlated with numbing and vmPFC activity was positively correlated with behavioral avoidance even though there were no functional group differences in this region of interest. Additionally, PTSD patients failed to appropriately reduce subjective arousal to the CS- over the course of the experiment and to the CS+ during extinction. Taken together, the results of study 1 support the hypothesis that PTSD is characterized by aberrant activity within regions of the neurocircuitry model, which leads to deficient extinction maintenance. Furthermore, our data confirm a general inability of PTSD patients to correctly identify safety signals and modulate fear responses based on this information. Such dysfunctional mechanisms seem to contribute to PTSD symptoms and represent a probable cause for relapse, whereas resilient subjects appear to benefit from protective mechanisms. In the second study, subjects underwent a resting state scan and functional connectivity was analyzed using an amygdala seed and independent component analysis (ICA) as well as correlations with symptom severity. The seed-based approach revealed increased left amygdala – the left insula coupling in PTSD versus nonPTSD, which positively correlated with re-experiencing intensity. Compared to HC, both trauma experienced groups showed higher positive correlations of the left amygdala and the right putamen as well as the right insula. The ICA did not reveal any group differences, i.e. in DMN connectivity. In summary, study 2 indicates that altered amygdala-insula coupling and decreased amygdala-putamen coupling, but not DMN connectivity, contribute to the pathophysiology of PTSD. Hyperconnectivity between the left amygdala and the left insula differentiated patients from resilient subjects and was linked to re-experiencing intensity. This result suggests that a stronger functional link between somatic sensations and emotional appraisal might lead to increased anticipation of negative events in PTSD, which potentially explains characteristic symptoms such as hyperarousal and negative alterations in mood and cognition

HIV-1 TAT1-86 INDUCED EFFECTS ON EXTINCTION AND RELAPSE IN FEAR CONDITIONING LEARNING

HIV-1 Associated Neurocognitive Disorder (HAND) is a neurodegenerative condition affecting roughly 30-50% of HIV-1 infected individuals. Symptoms include a wide range of cognitive impairments, but this series of experiments focuses on deficits in learning and memory. Specifically, these experiments investigate behavioral deficits in associative learning using fear conditioning methodology. The transactivator of transcription (Tat), In humans, the severity of symptoms is most strongly correlated with the severity of synaptic disruption and dendritic injury. Of the HIV-1 viral proteins, Tat plays a key role in facilitating structural and functional dendritic defects in neurons. Within the brain, Tat has several direct and indirect effects that result in structural and functional changes to regions of the brain crucial for associative learning. Fear conditioning using a Tat transgenic mouse model of HAND allows for the study of these affected regions while minimizing the influence of motivational systems. In fear conditioning, subjects are presented with a conditioned stimulus (CS) followed by unconditioned stimulus (US). The subject learns an association between the two, and performs a conditioned response (CR), indicative of learning, in preparation for the US. The acquisition of this response can then undergo extinction in which the US is presented multiple times without the US, thus reducing the conditioned response. Finally, relapse in conditioned responding can be observed due to changes in context, and reminders of the US. There are regionspecific contributions to each of these learning processes, allowing for the connection between specific deficits and associated brain regions. The current series of experiments investigated acquisition, extinction, and relapse of conditioned responding.Results from these experiments reveal transient deficits in acquisition for male and female Tat(+) subjects and a transient deficit in extinction learning seen only in male Tat(+) subjects. These findings indicate disruption to amygdala and prefrontal cortex circuitry. Renewal, a contextual form of relapse, was not observed while reinstatement, a US reminder form of relapse, was. These results indicate that the failure to observe renewal were not due to failure to recall acquisition memories. Overall, these experiments establish clear methodology for investigating associative learning deficits in the Tat transgenic mouse model of HAND and demonstrate transient deficits related to acquisition and extinction.Doctor of Philosoph

The Role of the Hippocampus in Representations of Emotional Memory

Although the hippocampus has long been implicated in contextual fear learning, the exact function of this brain structure is unclear. It is generally thought to encode a spatial context with which a fear memory can be associated, but how it may accomplish this and whether it plays a role in emotional memory is largely unknown. It is also unclear whether the hippocampus acts as a single unitary structure, or whether the dorsal and ventral poles, which exhibit differential connectivity to other brain regions, function independently. This dissertation examines the involvement of the hippocampus in emotional learning. A contextual fear conditioning paradigm using a predator odor as an ethologically relevant fearful stimulus was developed and lesions and immunohistochemistry were used to examine differential involvement of the dorsal and ventral hippocampus in response to fear learning. Long-term physiological recordings of dorsal place cells were then conducted to determine the effects of fear conditioning and also fear extinction on contextual representations in the hippocampus. Additionally, cells in the ventral hippocampus were assessed for responses to visuospatial manipulations and changing odor cues of varying emotional valence. It was found that the dorsal and ventral hippocampal regions are both independently required for contextual fear conditioning, and neurons in each region are differentially activated in response to fear learning. Furthermore, place cells in the dorsal hippocampus remapped in response to fear conditioning and stabilized those new fields in the long term. Extinction training caused many place cells to remap once again, suggesting that the dorsal hippocampus encodes varying representations of `fearful\u27 and `safe\u27 contexts. Finally, cells in the ventral hippocampus exhibited stronger responses to anxiogenic contextual cues compared to dorsal cells. In conclusion, these data suggest that the hippocampus is involved in emotional learning and that its function may vary along its longitudinal axis

The Psychological and Neural Bases of Extinction Learning

Extinction is a fundamental learning and memory process that enables humans and animals to survive in the face of shifting environmental conditions. The context-specific nature of extinction learning is demonstrated by the renewal phenomenon, in which responding returns following a change in context after extinction. Pavlovian fear conditioning procedures have primarily been used to investigate the psychological and neural processes mediating extinction. However, compared to passive defensive strategies, the mechanisms governing the extinction of active defensive strategies are not well understood. This thesis examined the psychological processes mediating the extinction of both active and passive defensive responses using the shock-probe defensive burying task. We found robust ABA renewal and less marked ABC and AAB renewal of passive coping behaviours. Active coping strategies linked to conditioned defensive burying did not display renewal, indicating that passive coping strategies are more prone to renewal than active coping strategies.These findings have important implications for understanding how context influences the extinction of different defensive responses to aversive stimuli. Moreover, this thesis employed an appetitive Pavlovian conditioning procedure to investigate the neural mechanisms mediating the extinction of responding to a discrete sucrose cue. Using Fos immunohistochemistry and correlational network analysis, we identified the neural correlates and networks associated with the recall vs extinction of responding to a sucrose-predictive Pavlovian cue. Our findings are consistent with those obtained using Pavlovian fear and operant reward-seeking procedures, which have demonstrated a functional dichotomy between the prelimbic (PL) and the infralimbic (IL) cortices of the medial prefrontal cortex. Namely, our results were consistent with the idea that the PL promotes the expression, while the IL mediates the extinction of conditioned responding. Additionally, we found that the paraventricular nucleus of the thalamus (PVT) plays a role in the recall of appetitive Pavlovian responding, and a neural network including the IL and PVT is active during extinction but not recall, suggesting that IL projections to the PVT may be involved in appetitive Pavlovian extinction. In support of this hypothesis, additional experiments found that optical stimulation of the IL-to-PVT pathway completely blocked appetitive Pavlovian renewal, while stimulation of the PL-to-PVT pathway had only modest effects on renewal. In the same experiments, stimulation of the IL-to-PVT, but not the PL-to-PVT, pathway supported self-stimulation, suggesting that this pathway has a reinforcing property. Together, these findings provide novel insights into the neural mechanisms underlying the extinction of responding to appetitive Pavlovian cues, and they point to the PVT as a critical node in the neural circuitry underlying the extinction of appetitive Pavlovian conditioned responding

Could Stress Contribute to Pain-Related Fear in Chronic Pain?

Learning to predict pain based on internal or external cues constitutes a fundamental and highly adaptive process aimed at self-protection. Pain-related fear is an essential component of this response, which is formed by associative and instrumental learning processes. In chronic pain, pain-related fear may become maladaptive, drive avoidance behaviors and contribute to symptom chronicity. Pavlovian fear conditioning has proven fruitful to elucidate associative learning and extinction involving aversive stimuli, including pain, but studies in chronic pain remain scarce. Stress demonstrably exerts differential effects on emotional learning and memory processes, but this has not been transferred to pain-related fear. Within this perspective, we propose that stress could contribute to impaired pain-related associative learning and extinction processes and call for interdisciplinary research. Specifically, we suggest to test the hypotheses that (1) extinction-related phenomena inducing a re-activation of maladaptive pain-related fear (e.g., reinstatement, renewal) likely occur in everyday life of chronic pain patients and may alter pain processing, impair perceptual discrimination and favour overgeneralization; (2) acute stress prior to or during acquisition of pain-related fear may facilitate the formation and/or consolidation of pain-related fear memories, (3) stress during or after extinction may impair extinction efficacy resulting in greater reinstatement or context-dependent renewal of pain-related fear; and (4) these effects could be amplified by chronic stress due to early adversity and/or psychiatric comorbidity such as depression or anxiety in patients with chronic pain