A randomized controlled trial of the effect of D-cycloserine on extinction and fear conditioning in humans

Previous research has shown that D-cycloserine (DCS) facilitates extinction of Pavlovian fear conditioning in rats and enhances exposure therapy in humans. The aim of this study was to test the effect of DCS on extinction of fear conditioning in humans. In three experiments, 238 participants were given either DCS (50 or 500 mg) or placebo 2-3 h before extinction training following a differential shock conditioning paradigm. Clear extinction and recovery (return of fear) effects were observed on both skin conductance and self-reported shock expectancy measures in three studies. DCS had no influence on these effects. The same pattern was observed when the analysis was restricted to aware participants or to good conditioners, when fear-relevant cues (pictures of snakes) were used as the conditioned stimuli, or when analysis was restricted to heightened snake-fearful participants. These results suggest that DCS may not enhance the extinction, or prevent the recovery, of learned fear in a differential Pavlovian conditioning paradigm in humans. Further experimental research is needed to better understand the mechanisms underlying the therapeutic effects of DCS.     

Enhancing exposure-based therapy from a translational research perspective

Combining an effective psychological treatment with conventional anxiolytic medication is typically not more effective than unimodal therapy for treating anxiety disorders. However, recent advances in the neuroscience of fear reduction have led to novel approaches for combining psychological therapy and pharmacological agents. Exposure-based treatments in humans partly rely on extinction to reduce the fear response in anxiety disorders. Animal studies have shown that D-cycloserine (DCS), a partial agonist at the glycine recognition site of the glutamatergic N-methyl-D-aspartate receptor facilitates extinction learning. Similarly, recent human trials have shown that DCS enhances fear reduction during exposure therapy of some anxiety disorders. This article discusses the biological and psychological mechanisms of extinction learning and the therapeutic value of DCS as an augmentation strategy for exposure therapy. Areas of future research will be identified.     

Effects of multiple exposures to D-cycloserine on extinction of conditioned fear in rats

Previous research has shown that an acute, post-training injection of D-cycloserine (DCS) facilitates extinction of conditioned fear in rats; however, the effects of multiple exposures to DCS in this situation are not known. In Experiment 1, rats were conditioned (light-shock pairings) and 24 h later given six extinction (light-alone) trials followed by an injection of DCS (15 mg/kg) or saline. The next day, all rats were tested for light-elicited freezing. In Experiment 2, the effect of DCS on extinction was tested in the same manner, except that rats were pre-exposed to DCS (0, 1, or 5 injections) just prior to conditioning. In Experiment 3, rats received five pre-exposures of DCS but conditioning occurred either 2 or 28 days after the last pre-exposure. The results showed that DCS facilitated extinction of conditioned freezing to the light CS when no drug pre-exposure had occurred, but pre-exposure to DCS just prior to conditioning disrupted the facilitation of extinction effect. When 28 days were interposed between pre-exposure and conditioning, the facilitatory effects of DCS on extinction were restored. These findings suggest that DCS has significant clinical value but that behavioral desensitization may occur with multiple exposures; however, desensitization is not permanent and is reduced by the passage of time.     

The effect of D-cycloserine on immediate vs. delayed extinction of learned fear

We compared the effect of D-cycloserine (DCS) on immediate (10 min after conditioning) and delayed (24 h after conditioning) extinction of learned fear in rats. DCS facilitated both immediate and delayed extinction when the drug was administered after extinction training. However, DCS did not facilitate immediate extinction when administered prior to extinction training (i.e., when the interval between drug administration and shock was reduced). In addition, administering five, but not two, shocks prior to extinction training disrupted the facilitating effects of DCS on delayed extinction. These results suggest that aversive experiences prior to DCS administration can prevent it from facilitating extinction.     

D-cycloserine facilitates context-specific fear extinction learning

D-cycloserine (DCS) may facilitate fear extinction learning, but the behavioral consequences and mechanisms behind this effect are not well understood at present. In this paper, we re-analyze data from previously reported null result experiments and find that rats showing above-median extinction learning during DCS treatment benefited from the drug, whereas rats showing below-median (and in this case little) extinction learning did not. Two additional experiments found that DCS facilitated extinction learning when specifically combined with a moderate, but not a small, number of extinction trials. DCS thus facilitates extinction learning only if the behavioral procedure first engages the extinction learning process. The benefits of the drug, however, were specific to the context in which extinction was learned--i.e., DCS did not prevent or influence the renewal of fear observed when the extinguished cue was tested in the original conditioning context.     

Single prolonged stress disrupts retention of extinguished fear in rats

Clinical research has linked post-traumatic stress disorder (PTSD) with deficits in fear extinction. However, it is not clear whether these deficits result from stress-related changes in the acquisition or retention of extinction or in the regulation of extinction memories by context, for example. In this study, we used the single prolonged stress (SPS) animal model of PTSD and fear conditioning procedures to examine the effects of prior traumatic stress on the acquisition, retention, and context-specificity of extinction. SPS administered one week prior to fear conditioning had no effect on the acquisition of fear conditioning or extinction but disrupted the retention of extinction memories for both contextual and cued fear. This SPS effect required a post-stress incubation period to manifest. The results demonstrate that SPS disrupts extinction retention, leading to enhanced fear renewal; further research is needed to identify the neurobiological processes through which SPS induces these effects.     

Effects of D-cycloserine on extinction of learned fear to an olfactory cue

D-cycloserine (DCS), a partial NMDA receptor agonist, facilitates extinction of learned fear in rats and has been used to treat anxiety disorders in clinical populations. However, research into the effects of DCS on extinction is still in its infancy, with visual cues being the primary fear-eliciting stimuli under investigation. In both human and animal subjects odors have been found to associate strongly with aversive events. Therefore, this study examined the generality of the effects of DCS on extinction by testing odor cues. Sprague-Dawley rats were conditioned and extinguished to an odor using varying parameters, injected with either saline or DCS (15 mg/kg) following extinction, and then tested for a freezing response 24 h later. Experiment 1 demonstrated that after 3 odor-shock pairings, rats did not display short-term extinction and DCS had no effect on long-term extinction. Experiment 2 demonstrated that after 3 odor-noise pairings, rats displayed significant short-term extinction and DCS significantly facilitated long-term extinction. Following 2 odor-shock pairings in Experiment 3, half the rats displayed short-term extinction ("extinguishers") and half did not ("non-extinguishers"). DCS facilitated long-term extinction in the "extinguishers" condition but not in the "non-extinguishers" condition. In Experiment 4, following 2 odor-shock pairings and an extra extinction session, DCS had a significant facilitatory effect on long-term extinction. Thus, extinction of freezing to an odor cue was facilitated by systemic injections of DCS, but only when some amount of within-session extinction occurred prior to injection.     

Preextinction Stress Prevents Context-Related Renewal of Fear

Extinction learning, which creates new safety associations, is thought to be the mechanism underlying exposure therapy, commonly used for the treatment of anxiety disorders and posttraumatic stress disorder. The relative strength and availability for retrieval of both the fear and safety memories determine the response in a given situation. While the fear memory is often context-independent and may easily generalize, extinction memory is highly context-specific. “Renewal” of the extinguished fear memory might thus occur following a shift in context. The aim of the current work was to create an enhanced and generalized extinction memory to a discrete stimulus using stress exposure before extinction learning, thereby preventing renewal. In our contextual fear conditioning paradigm, 40 healthy men acquired (Day 1), retrieved and extinguished (Day 2) the fear memories, with no differences between the stress and the control group. A significant difference between the groups emerged in the renewal test (Day 3). A renewal effect was seen in the control group (N = 20), confirming the context-dependency of the extinction memory. In contrast, the stress group (N = 20) showed no renewal effect. Fear reduction was generalized to the acquisition context as well, suggesting that stress rendered the extinction memory more context-independent. These results are in line with previous studies that showed contextualization disruption as a result of pre-learning stress, mediated by the rapid effects of glucocorticoids on the hippocampus. Our findings support research investigating the use of glucocorticoids or stress induction in exposure therapy and suggest the right timing of administration in order to optimize their effects.     

Polyaminergic agents modulate contextual fear extinction in rats

Polyamines, such as spermidine and spermine, have been reported to improve memory retention through the activation of N-methyl-d-aspartate receptors (NMDAr). However whether polyamine agonists and antagonists alter extinction remains unclear. In the current study, we investigated whether spermidine and polyamine antagonists that selectively block the NR2B subunit at the NMDAr alter the extinction of contextual conditioned fear in male Wistar rats. The bilateral intra-hippocampal administration of exogenous spermidine (2 nmol/site) immediately after, but not 6 h after extinction training, facilitated the extinction of fear conditioning. The injection of the NMDAr antagonists arcaine (0.2 nmol/site), ifenprodil (20 nmol/site) and traxoprodil (0.2 nmol/site), disrupted fear extinction and, at doses that had no effect per se, reversed the facilitatory effect of spermidine on fear extinction. These results suggest that exogenous and endogenous polyamines facilitate the extinction of contextual conditioned fear through activation of NR2B subunit-containing NMDAr in the hippocampus. Since extinction-based exposure therapy is widely used as treatment for a number of anxiety-related disorders, including phobias and post-traumatic stress, the currently reported facilitation of extinction by polyaminergic agents suggest these compounds as putative candidates for drug development.     

Facilitation of fear extinction by D-cycloserine: theoretical and clinical implications

Anxiety disorders are among the most common psychological disturbances in the industrialized world. Current behavioral therapy procedures for these disorders are somewhat effective, but their efficacy could be substantially improved. Because these procedures are largely based on the process of extinction, manipulations that enhance extinction may lead to improvements in treatment effectiveness. We review the evidence that D-cycloserine (DCS), a partial NMDA agonist, facilitates extinction of learned fear in rats. Although only a few studies have examined the effects of DCS on extinction of learned fear, this work suggests that this drug may have a number of potential clinical benefits. In addition, attempts at interpreting this research illustrate our limited understanding of the processes involved in extinction.     

Inhibition of PKA anchoring to A-kinase anchoring proteins impairs consolidation and facilitates extinction of contextual fear memories

Both genetic and pharmacological studies demonstrated that contextual fear conditioning is critically regulated by cyclic AMP-dependent protein kinase (PKA). Since PKA is a broad range protein kinase, a mechanism for confining its activity is required. It has been shown that intracellular spatial compartmentalization of PKA signaling is mediated by A-kinase anchoring proteins (AKAPs). Here, we investigated the role of PKA anchoring to AKAPs in different stages of the memory process (acquisition, consolidation, retrieval and extinction) using contextual fear conditioning, a hippocampus-dependent learning task. Mice were injected intracerebroventricularly or intrahippocampally with the membrane permeable PKA anchoring disrupting peptides St-Ht31 or St-superAKAP-IS at different time points during the memory process. Blocking PKA anchoring to AKAPs resulted in an impairment of fear memory consolidation. Moreover, disrupted PKA anchoring promoted contextual fear extinction in the mouse hippocampus. We conclude that the temporal and spatial compartmentalization of hippocampal PKA signaling pathways, as achieved by anchoring of PKA to AKAPs, is specifically instrumental in long-term contextual fear memory consolidation and extinction, but not in acquisition and retrieval.     

Recalling safety: cooperative functions of the ventromedial prefrontal cortex and the hippocampus in extinction

Anxiety disorders are commonly treated with exposure-based therapies that rely on extinction of conditioned fear. Persistent fear and anxiety following exposure therapy could reflect a deficit in the recall of extinction learning. Animal models of fear learning have elucidated a neural circuit for extinction learning and recall that includes the amygdala, ventromedial prefrontal cortex (vmPFC), and hippocampus. Whereas the amygdala is important for extinction learning, the vmPFC is a site of neural plasticity that allows for the inhibition of fear during extinction recall. We suggest that the vmPFC receives convergent information from other brain regions, such as contextual information from the hippocampus, to determine the circumstances under which extinction or fear will be recalled. Imaging studies of human fear conditioning and extinction lend credence to this extinction network. Understanding the neural circuitry underlying extinction recall will lead to more effective therapies for disorders of fear and anxiety.     

Contribution of Ventrolateral Prefrontal Cortex to the Acquisition and Extinction of Conditioned Fear in Rats

The ventrolateral, agranular insular portion of prefrontal cortex (PFC) in rats is involved in visceral functions and has been shown to be involved in emotional processes. However, its contribution to aversive learning has not been well defined. Classical fear conditioning has been a powerful tool for illuminating some of the primary neural structures involved in aversive emotional learning. We measured both the acquisition and the extinction of conditioned fear following lesions of the ventrolateral PFC of rats. Lesions reduced fear reactivity to contextual stimuli associated with conditioning without affecting CS acquisition, and had no effect on response extinction. Ventrolateral PFC may normally be involved in the processing of contextual information while not being directly involved in extinction processes within the aversive domain.     

睡眠对恐惧学习的影响及其认知神经机制

睡眠问题可能会诱发恐惧相关情绪障碍(焦虑、创伤性应激障碍、恐怖症等),研究睡眠影响恐惧学习的认知神经机制,有助于增强对恐惧相关情绪障碍的预测、诊断和治疗。以往研究表明睡眠剥夺影响恐惧习得和消退主要是通过抑制vmPFC活动,阻碍其与杏仁核的功能连接,从而导致恐惧习得增强或是消退学习受损。进一步研究发现睡眠不同阶段对恐惧学习相关脑区有独特的影响:剥夺(缺乏)快速眼动睡眠会抑制vmPFC活动、增强杏仁核、海马激活,导致恐惧习得增强,消退学习受损,此外边缘皮层的功能连接减少破坏了记忆巩固(恐惧记忆和消退记忆);而慢波睡眠主要与海马变化有关,慢波睡眠期间进行目标记忆重激活可促进恐惧消退学习。未来研究需要增加睡眠影响恐惧泛化的神经机制研究、及昼夜节律中断对恐惧消退的影响,以及关注动物睡眠研究向人类睡眠研究转化中存在的问题。     

Transgenic inhibition of neuronal calcineurin activity in the forebrain facilitates fear conditioning, but inhibits the extinction of contextual fear memories

It is unclear whether protein phosphatases, which counteract the actions of protein kinases, play a beneficial role in the formation and extinction of previously acquired fear memories. In this study, we investigated the role of the calcium/calmodulin dependent phosphatase 2B, also known as calcineurin (CaN) in the formation of contextual fear memory and extinction of previously acquired contextual fear. We used a temporally regulated transgenic approach, that allowed us to selectively inhibit neuronal CaN activity in the forebrain either during conditioning or only during extinction training leaving the conditioning undisturbed. Reducing CaN activity through the expression of a CaN inhibitor facilitated contextual fear conditioning, while it impaired the extinction of previously formed contextual fear memory. These findings give the first genetic evidence that neuronal CaN plays an opposite role in the formation of contextual fear memories and the extinction of previously formed contextual fear memories.     

The Effect of d-Cycloserine on Subliminal Cue Exposure in Spider Fearful Individuals

Research on d-cycloserine (DCS) has demonstrated a significant effect on symptom reduction in human studies that utilized conventional exposure-based approaches. Recent studies have offered promising results for targeting fears through subliminal paradigms. In this double-blind, randomized placebo-controlled study, 45 spider fearful individuals received DCS or placebo pills prior to completing a subliminal cue exposure task to images of spiders. Participants completed self-report questionnaires and a behavioral approach task to a live caged tarantula. After repeated exposure to subliminal spider cues, participants in the DCS group reported a greater reduction in disgust than individuals in the placebo group. No difference was observed in fear ratings. These findings suggest that DCS augments the reduction in disgust in spider fearful subjects after subliminal exposure to spider cues.     

Factors regulating the effects of hippocampal inactivation on renewal of conditional fear after extinction

After extinction of fear to a Pavlovian conditional stimulus (CS), contextual stimuli come to regulate the expression of fear to that CS. There is growing evidence that the context dependence of memory retrieval after extinction involves the hippocampus. In the present experiment, we examine whether hippocampal involvement in memory retrieval after extinction is related to the history of CS presentations in the context used for retrieval testing. We used infusions of muscimol to inactivate the dorsal hippocampus (DH) during postextinction retrieval tests that were conducted in contexts that differed in their history of CS presentations in that context. We found that DH inactivation affected the context-dependent retrieval of extinction (i.e., renewal) when testing occurred in a context that had no history of CS exposure, but not in a context that reliably predicted the CS. These results are discussed in terms of theories regarding the role of the hippocampus in contextual memory retrieval.     

Methylphenidate enhances extinction of contextual fear

Methylphenidate (MPH, Ritalin) is a norepinephrine and dopamine transporter blocker that is widely used in humans for treatment of attention deficit disorder and narcolepsy. Although there is some evidence that targeted microinjections of MPH may enhance fear acquisition, little is known about the effect of MPH on fear extinction. Here, we show that MPH, administered before or immediately following extinction of contextual fear, will enhance extinction retention in C57BL/6 mice. Animals that received MPH (2.5-10 mg/kg) before an extinction session showed decreased freezing response during extinction, and the effect of the 10 mg/kg dose on freezing persisted to the next day. When MPH (2.5-40 mg/kg) was administered immediately following an extinction session, mice that received MPH showed dose-dependent decreases in freezing during subsequent tests. MPH administered immediately after a 3-min extinction session or 4 h following the first extinction session did not cause significant differences in freezing. Together, these findings demonstrate that MPH can enhance extinction of fear and that this effect is sensitive to dose, time of injection, and duration of the extinction session. Because MPH is widely used in clinical treatments, these experiments suggest that the drug could be used in combination with behavioral therapies for patients with fear disorders.     

Extinction partially reverts structural changes associated with remote fear memory

Structural synaptic changes occur in medial prefrontal cortex circuits during remote memory formation. Whether extinction reverts or further reshapes these circuits is, however, unknown. Here we show that the number and the size of spines were enhanced in anterior cingulate (aCC) and infralimbic (ILC) cortices 36 d following contextual fear conditioning. Upon extinction, aCC spine density returned to baseline, but the enhanced proportion of large spines did not. Differently, ILC spine density remained elevated, but the size of spines decreased dramatically. Thus, extinction partially erases the remote memory network, suggesting that the preserved network properties might sustain reactivation of extinguished conditioned fear.     

Naloxone and Pavlovian fear conditioning

Previous research has shown that pretreating rats with the opiate antagonist naloxone increases the freezing that follows painful electric shock. Three experiments, using freezing behavior as a dependent variable, were carried out to determine whether the drug might cause this effect by enhancing fear conditioning. Two of the studies employed a differential context fear-conditioning paradigm. Naloxone did not affect freezing behavior during the preshock adaptation period. In Experiment 1, naloxone was found to increase resistance to extinction in the S+ context. In Experiment 2, naloxone was found to increase freezing in the S+ context. This effect was dependent upon administering naloxone during training but not dependent on administering it during testing. The third study employed a generalization paradigm. It was found that naloxone's effect on postshock freezing was dependent on the place of testing; as the contextual cues of the test chamber were changed from those of the conditioning chamber, the effect of naloxone on freezing was reduced. The results of these experiments lend strong support to the hypothesis that naloxone increases freezing by enhancing the conditioning of fear to contextual stimuli associated with shock.