High Trait Anxiety: A Challenge for Disrupting Fear Memory Reconsolidation

Disrupting reconsolidation may be promising in the treatment of anxiety disorders but the fear-reducing effects are thus far solely demonstrated in the average organism. A relevant question is whether disrupting fear memory reconsolidation is less effective in individuals who are vulnerable to develop an anxiety disorder. By collapsing data from six previous human fear conditioning studies we tested whether trait anxiety was related to the fear-reducing effects of a pharmacological agent targeting the process of memory reconsolidation - n = 107. Testing included different phases across three consecutive days each separated by 24 h. Fear responding was measured by the eye-blink startle reflex. Disrupting the process of fear memory reconsolidation was manipulated by administering the β-adrenergic receptor antagonist propranolol HCl either before or after memory retrieval. Trait anxiety uniquely predicted the fear-reducing effects of disrupting memory reconsolidation: the higher the trait anxiety, the less fear reduction. Vulnerable individuals with the propensity to develop anxiety disorders may need higher dosages of propranolol HCl or more retrieval trials for targeting and changing fear memory. Our finding clearly demonstrates that we cannot simply translate observations from fundamental research on fear reduction in the average organism to clinical practice.     

Social Isolation Stress-Induced Fear Memory Deficit is Mediated by Down-Regulated Neuro-Signaling System and Egr-1 Expression in the Brain

We previously reported that social isolation (SI) rearing of rodents not only elicits a variety of behavioral abnormalities including attention deficit hyperactivity disorder-like behaviors, but also impairs fear memory in mice. This study aimed to clarify a putative mechanism underlying SI-induced conditioned fear memory deficit. Mice were group-housed (GH) or socially isolated for 2 weeks or more before the experiments. SI animals acquired contextual and auditory fear memory elucidated at 90 min and 4 h after training, respectively; however, they showed significantly impaired contextual and auditory memory performance at 24 h and 4 days after the training, respectively, indicating SI-induced deficit of the consolidation process of fear memory. Neurochemical studies conducted after behavioral tests revealed that SI mice had a significantly down-regulated level of Egr-1 but not Egr-2 in the hippocampal and cortical cytosolic fractions compared with those levels in the GH control animals. Moreover, in the SI group, phosphorylated levels of synaptic plasticity-related signaling proteins in the hippocampus, NR1 subunit of N-methyl-d-aspartate receptor, glutamate receptor 1, and calmodulin-dependent kinase II but not cyclic AMP-responsive element binding protein were significantly down-regulated compared with those levels in GH animals, whereas non-phosphorylated levels of these proteins were not affected by SI. These findings suggest that dysfunctions of Egr-1 and neuro-signaling systems are involved in SI-induced deficits of fear memory consolidation in mice.     

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone^1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed³. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory⁴. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface^3,4.Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference^5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later^5,11,16, although this may vary depending on the strength and age of the memory¹⁵. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates^10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies^12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters¹¹. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor¹⁶, episodic¹⁷, or declarative¹⁸ memories leads to interference with the original memory trace¹⁴. We outline below a novel protocol used to block fear recovery in humans.     

Mapping and Deciphering Neural Codes of NMDA Receptor-Dependent Fear Memory Engrams in the Hippocampus

Mapping and decoding brain activity patterns underlying learning and memory represents both great interest and immense challenge. At present, very little is known regarding many of the very basic questions regarding the neural codes of memory: are fear memories retrieved during the freezing state or non-freezing state of the animals? How do individual memory traces give arise to a holistic, real-time associative memory engram? How are memory codes regulated by synaptic plasticity? Here, by applying high-density electrode arrays and dimensionality-reduction decoding algorithms, we investigate hippocampal CA1 activity patterns of trace fear conditioning memory code in inducible NMDA receptor knockout mice and their control littermates. Our analyses showed that the conditioned tone (CS) and unconditioned foot-shock (US) can evoke hippocampal ensemble responses in control and mutant mice. Yet, temporal formats and contents of CA1 fear memory engrams differ significantly between the genotypes. The mutant mice with disabled NMDA receptor plasticity failed to generate CS-to-US or US-to-CS associative memory traces. Moreover, the mutant CA1 region lacked memory traces for “what at when” information that predicts the timing relationship between the conditioned tone and the foot shock. The degraded associative fear memory engram is further manifested in its lack of intertwined and alternating temporal association between CS and US memory traces that are characteristic to the holistic memory recall in the wild-type animals. Therefore, our study has decoded real-time memory contents, timing relationship between CS and US, and temporal organizing patterns of fear memory engrams and demonstrated how hippocampal memory codes are regulated by NMDA receptor synaptic plasticity.     

Worrying affects associative fear learning: a startle fear conditioning study

A valuable experimental model for the pathogenesis of anxiety disorders is that they originate from a learned association between an intrinsically non-aversive event (Conditioned Stimulus, CS) and an anticipated disaster (Unconditioned Stimulus, UCS). Most anxiety disorders, however, do not evolve from a traumatic experience. Insights from neuroscience show that memory can be modified post-learning, which may elucidate how pathological fear can develop after relatively mild aversive events. Worrying--a process frequently observed in anxiety disorders--is a potential candidate to strengthen the formation of fear memory after learning. Here we tested in a discriminative fear conditioning procedure whether worry strengthens associative fear memory. Participants were randomly assigned to either a Worry (n = 23) or Control condition (n = 25). After fear acquisition, the participants in the Worry condition processed six worrisome questions regarding the personal aversive consequences of an electric stimulus (UCS), whereas the Control condition received difficult but neutral questions. Subsequently, extinction, reinstatement and re-extinction of fear were tested. Conditioned responding was measured by fear-potentiated startle (FPS), skin conductance (SCR) and UCS expectancy ratings. Our main results demonstrate that worrying resulted in increased fear responses (FPS) to both the feared stimulus (CS(+)) and the originally safe stimulus (CS(-)), whereas FPS remained unchanged in the Control condition. In addition, worrying impaired both extinction and re-extinction learning of UCS expectancy. The implication of our findings is that they show how worry may contribute to the development of anxiety disorders by affecting associative fear learning.     

Effect of early androgen treatment on subsequent social behavior in heifers

Two groups of four heifers received daily injections of 0.30 mg/kg of testosterone propionate in oil for 100 days, between 3 and 6 months of age. Two other groups of four heifers of the same age received only oil and served as controls. Three to five months after the end of treatment, a series of experiments was designed to compare dominance ability, aggressiveness, and reactions associated with fear, between treated and control animals. Dominance ability, when faced with animals of the same age and social experience, is higher for treated animals than controls, 85% of them being dominant. Treated animals established dominance relationships between themselves more slowly than did control animals or mixed pairs. In all experiments treated animals fought more often than controls. Those which became subordinate fought more than those which became dominant. When subordinate, treated animals withdrew less often than control animals and frequently did not react to aggression. When dominant, treated animals were never more, and sometimes less, aggressive than controls. It is proposed that changes in social structure of organized groups and the capacity to become dominant at first encounter are due to a reduction of fear of other animals induced by androgens. The acquisition of social rank in cattle may be principally due to differences in fear or “emotionality” rather than in aggressiveness.     

Serum- and glucocorticoid-inducible kinase1 enhances contextual fear memory formation through down-regulation of the expression of Hes5

Mitogen-activated protein kinase/extracellular signal-regulated kinase plays an important role in memory formation and directly phosphorylates serum- and glucocorticoid-inducible kinase1 (SGK1) at Ser78. In this study, we examined the role and mechanism of SGK1 Ser78 activation involved in contextual fear memory formation in rats. Results revealed that SGK1 Ser78 phosphorylation was increased 30 min, 1 h and 3 h after training. Transient transfection of the dominant negative mutant of sgk, sgkS78A, to hippocampal neurons impaired, whereas transfection of the constitutively active sgk, sgkS78D, enhanced fear retention. Microarray analysis identified 14 genes that showed more than threefold alteration in their gene expression in sgkS78A-transfected animals 6 h after training. One of them is Hairy and Enhancer of split 5 (Hes5). The expression level of Hes5 is approximately 4.4-fold higher in sgkS78A-transfected animals. Further analyses revealed that Hes5 level is markedly decreased after training in control animals, but sgkS78A markedly increased Hes5 level after training. RNA interference experiment showed that shHes5 dose-dependently enhanced fear retention, whereas over-expression of Hes5 impaired fear retention. Moreover, shHes5 at a lower concentration completely blocked the memory-impairing effect of sgkS78A. These results together suggest that Hes5 negatively regulates contextual fear memory formation and SGK activation down-regulates Hes5 expression to enhance fear retention.     

In vitro synaptic reconsolidation in amygdala slices prepared from rat brains

The retrieval of consolidated fear memory causes it to be labile or deconsolidated, and the deconsolidated fear memory is reconsolidated over time in a protein synthesis-dependent manner. We have recently developed an ex vivo model where during fear memory deconsolidation and reconsolidation the synaptic state can be monitored at thalamic input synapses onto the lateral amygdala (T-LA synapses), a storage site for auditory fear memory. In this ex vivo model, the deconsolidation and reconsolidation processes of auditory fear memory in the intact brain were prevented following brain slicing; therefore, we could monitor the synaptic state for memory deconsolidation and reconsolidation at the time of brain slicing. However, why the synaptic reconsolidation process stopped after brain slicing in the ex vivo model is not known. One possibility is that brain slicing severs neuromodulatory innervations, which are required for memory reconsolidation, from other brain regions (e.g., noradrenergic innervation). In the present study, we supplemented amygdala slices with exogenous norepinephrine as a substitute for the severed noradrenergic innervations. DHPG (a group I metabotropic glutamate receptor agonist)-induced depotentiation (mGluRI-depotentiation), a marker for consolidated synapses, was observed following norepinephrine application to slices prepared immediately after tone presentation (fear memory retrieval) to rats that had been pre-conditioned to a tone paired with a shock. These results suggest that noradrenergic activation initiates synaptic reconsolidation. In contrast, mGluRI-depotentiation was absent following norepinephrine application to slices that were prepared immediately after the tone presentation (no fear memory retrieval) to rats when a tone and a shock were unpaired, ruling out the possibility that noradrenergic activation somehow facilitates a subsequent synaptic depression induced by DHPG irrespective of synaptic reconsolidation. Furthermore, the restored mGluRI-depotentiation following application of exogenous norepinephrine was dependent on de novo protein synthesis, as is memory reconsolidation. Thus, our findings suggest that T-LA synapses from acute slice preparations can undergo a reconsolidation process, thereby providing an optimal preparation to study a fear memory reconsolidation process in vitro.     

Cortex- and Amygdala-Dependent Learning and Nicotinic Acetylcholine Receptor Gene Expression is Severely Impaired in Mice Orally Treated with AlCl<Subscript>3</Subscript>

Recent industrialization has increased human exposure to bio-available aluminum (Al). If more Al enters the brain than leaves, Al concentration will rise in the brain leading to neurodegenerative disorders. The aim of the present study was to determine Al concentration, neurodegeneration, and nicotinic acetylcholine receptor (nAChR) gene expression in the cortex and amygdala after oral ingestion of Al salt. The effect of Al on cortex- and amygdala-dependent learning and memory functions was also assessed. Mice were given AlCl₃ (250 mg/kg) in drinking water for 42 days. nAChR gene expression was determined in the cortex and amygdala. The mice were subjected to behavior tests (fear conditioning, fear extinction, and open field), to assess memory deficits. The acquisition of fear memory in the fear conditioning test remained unaffected due to the Al administration. However, fear extinction (which is a new learning) was severely impaired. The behavioral analysis in the open field test showed greater anxiety and less adaptability to the new environment in Al-treated animals. High Al concentration and severe neurodegeneration in the cortex were observed following Al treatment while a slight, non-significant elevation in Al concentration was observed in the amygdala of Al-treated animals. The analysis of nAChR gene expression via RT-PCR showed a significant reduction in expression of α7, α4, and β2 nAChR genes in the cortex of Al-treated animals, while in the amygdala, the level of the α4 nAChR gene remained unaltered. Oral Al ingestion causes neuropathological changes and suppresses expression of nAChR genes that lead to deficits in learning and higher anxiety in Al-treated animals.     

Inception of a false memory by optogenetic manipulation of a hippocampal memory engram

Memories can be easily distorted, and a lack of relevant animal models has largely hindered our understanding of false-memory formation. Here, we first identified a population of cells in the dentate gyrus (DG) of the hippocampus that bear the engrams for a specific context; these cells were naturally activated during the encoding phase of fear conditioning and their artificial reactivation using optogenetics in an unrelated context was sufficient for inducing the fear memory specific to the conditioned context. In a further study, DG or CA1 neurons activated by exposure to a particular context were labelled with channelrhodopsin-2 (ChR2). These neurons were later optically reactivated during fear conditioning in a different context. The DG experimental group showed increased freezing in the original context in which a foot shock was never delivered. The recall of this false memory was context specific, activated similar downstream regions engaged during natural fear-memory recall, and was also capable of driving an active fear response. Together, our data demonstrate that by substituting a natural conditioned stimulus with optogenetically reactivated DG cells that bear contextual memory engrams, it is possible to incept an internally and behaviourally represented false fear memory.     

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.     

Artificial Theta Stimulation Impairs Encoding of Contextual Fear Memory

Several experiments have demonstrated an intimate relationship between hippocampal theta rhythm (4–12 Hz) and memory. Lesioning the medial septum or fimbria-fornix, a fiber track connecting the hippocampus and the medial septum, abolishes the theta rhythm and results in a severe impairment in declarative memory. To assess whether there is a causal relationship between hippocampal theta and memory formation we investigated whether restoration of hippocampal theta by electrical stimulation during the encoding phase also restores fimbria-fornix lesion induced memory deficit in rats in the fear conditioning paradigm. Male Wistar rats underwent sham or fimbria-fornix lesion operation. Stimulation electrodes were implanted in the ventral hippocampal commissure and recording electrodes in the septal hippocampus. Artificial theta stimulation of 8 Hz was delivered during 3-min free exploration of the test cage in half of the rats before aversive conditioning with three foot shocks during 2 min. Memory was assessed by total freezing time in the same environment 24 h and 28 h after fear conditioning, and in an intervening test session in a different context. As expected, fimbria-fornix lesion impaired fear memory and dramatically attenuated hippocampal theta power. Artificial theta stimulation produced continuous theta oscillations that were almost similar to endogenous theta rhythm in amplitude and frequency. However, contrary to our predictions, artificial theta stimulation impaired conditioned fear response in both sham and fimbria-fornix lesioned animals. These data suggest that restoration of theta oscillation per se is not sufficient to support memory encoding after fimbria-fornix lesion and that universal theta oscillation in the hippocampus with a fixed frequency may actually impair memory.     

Behavioral interventions to eliminate fear responses

Fear memory underlies anxiety-related disorders, including posttraumatic stress disorder(PTSD). PTSD is a fear-based disorder,characterized by difficulties in extinguishing the learned fear response and maintaining extinction. Currently, the first-line treatment for PTSD is exposure therapy, which forms an extinction memory to compete with the original fear memory. However,the extinguished fear often returns under numerous circumstances, suggesting that novel methods are needed to eliminate fear memory or facilitate extinction memory. This review discusses research that targeted extinction and reconsolidation to manipulate fear memory. Recent studies indicate that sleep is an active state that can regulate memory processes. We also discuss the influence of sleep on fear memory. For each manipulation, we briefly summarize the neural mechanisms that have been identified in human studies. Finally, we highlight potential limitations and future directions in the field to better translate existing interventions to clinical settings.     

Interoceptive Focus Shapes the Experience of Time

The perception of time is a fundamental part of human experience. Recent research suggests that the experience of time emerges from emotional and interoceptive (bodily) states as processed in the insular cortex. Whether there is an interaction between the conscious awareness of interoceptive states and time distortions induced by emotions has rarely been investigated so far. We aimed to address this question by the use of a retrospective time estimation task comparing two groups of participants. One group had a focus on interoceptive states and one had a focus on exteroceptive information while watching film clips depicting fear, amusement and neutral content. Main results were that attention to interoceptive processes significantly affected subjective time experience. Fear was accompanied with subjective time dilation that was more pronounced in the group with interoceptive focus, while amusement led to a quicker passage of time which was also increased by interoceptive focus. We conclude that retrospective temporal distortions are directly influenced by attention to bodily responses. These effects might crucially interact with arousal levels. Sympathetic nervous system activation affecting memory build-up might be the decisive factor influencing retrospective time judgments. Our data substantially extend former research findings underscoring the relevance of interoception for the effects of emotional states on subjective time experience.     

A NMDA Receptor Antagonist,MK-801 Impairs Consolidating Extinction of Auditory Conditioned Fear Responses in a Pavlovian Model

Background

In auditory fear conditioning, repeated presentation of the tone in the absence of shock leads to extinction of the acquired fear responses. The glutamate N-methyl-D-aspartate receptor (NMDAR) is thought to be involved in the extinction of the conditioned fear responses, but its detailed role in initiating and consolidating or maintaining the fear extinction memory is unclear. Here we investigated this issue by using a NMDAR antagonist, MK-801.

Methods/Main Findings

The effects of immediate (beginning at 10 min after the conditioning) and delayed (beginning at 24 h after conditioning) extinctions were first compared with the finding that delayed extinction caused a better and long-lasting (still significant on the 20^th day after extinction) depression on the conditioned fear responses. In a second experiment, MK-801 was intraperitoneally (i.p.) injected at 40 min before, 4 h or 12 h after the delayed extinction, corresponding to critical time points for initiating, consolidating or maintaining the fear extinction memory. i.p. injection of MK-801 at either 40 min before or 4 h after delayed extinction resulted in an impairment of initiating and consolidating fear extinction memory, which caused a long lasting increased freezing score that was still significant on the 7th day after extinction, compared with extinction group. However, MK-801 administered at 12 h after the delayed extinction, when robust consolidation has been occurred and stabilized, did not affect the established extinction memory. Furthermore, the changed freezing behaviors was not due to an alteration in general anxiety levels, since MK-801 treatment had no effect on the percentage of open-arm time or open-arm entries in an Elevated Plus Maze (EPM) task.

Conclusions/Significance

Our data suggested that the activation of NMDARs plays important role in initiation and consolidation but not maintenance of fear extinction memory. Together with the fact that NMDA receptor is very important for memory, our data added experimental evidence to the concept that the extinction of conditioned fear responses is a procedure of initiating and consolidating new memory other than simply “erasing” the fear memory.     

Age-related deficits in neuronal physiology and cognitive function are recapitulated in young mice overexpressing the L-type calcium channel,CaV1.3

The calcium dysregulation hypothesis of brain aging posits that an age-related increase in neuronal calcium concentration is responsible for alterations in a variety of cellular processes that ultimately result in learning and memory deficits in aged individuals. We previously generated a novel transgenic mouse line, in which expression of the L-type voltage-gated calcium, Ca_V1.3, is increased by ~50% over wild-type littermates. Here, we show that, in young mice, this increase is sufficient to drive changes in neuronal physiology and cognitive function similar to those observed in aged animals. Specifically, there is an increase in the magnitude of the postburst afterhyperpolarization, a deficit in spatial learning and memory (assessed by the Morris water maze), a deficit in recognition memory (assessed in novel object recognition), and an overgeneralization of fear to novel contexts (assessed by contextual fear conditioning). While overexpression of Ca_V1.3 recapitulated these key aspects of brain aging, it did not produce alterations in action potential firing rates, basal synaptic communication, or spine number/density. Taken together, these results suggest that increased expression of Ca_V1.3 in the aged brain is a crucial factor that acts in concert with age-related changes in other processes to produce the full complement of structural, functional, and behavioral outcomes that are characteristic of aged animals.     

Aromatized testosterone attenuates contextual generalization of fear in male rats

Generalization is a common symptom of many anxiety disorders, and females are 60% more likely to suffer from an anxiety disorder than males. We have previously demonstrated that female rats display significantly accelerated rates of contextual fear generalization compared to male rats; a process driven, in part, by activation of ERβ. The current study was designed to determine the impact of estrogens on contextual fear generalization in male rats. For experiment 1, adult male rats were gonadectomized (GDX) and implanted with a capsule containing testosterone proprionate, estradiol, dihydrotestosterone proprionate (DHT), or an empty capsule. Treatment with testosterone or estradiol maintained memory precision when rats were tested in a different (neutral) context 1 day after training. However, male rats treated with DHT or empty capsules displayed significant levels of fear generalization, exhibiting high levels of fear in the neutral context. In Experiment 2, we used acute injections of gonadal hormones at a time known to elicit fear generalization in female rats (e.g. 24 h before testing). Injection treatment followed the same pattern of results seen in Experiment 1. Finally, animals given daily injections of the aromatase inhibitor, Fadrozole, displayed significant fear generalization. These data suggest that testosterone attenuates fear generalization likely through the aromatization testosterone into estradiol as animals treated with the non-aromatizable androgen, DHT, or animals treated with Fadrozole, displayed significant generalized fear. Overall, these results demonstrate a sex-dependent effect of estradiol on the generalization of contextual fear.     

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.     

Genetic disruptions of Drosophila Pavlovian learning leave extinction learning intact

Individuals who experience traumatic events may develop persistent posttraumatic stress disorder. Patients with this disorder are commonly treated with exposure therapy, which has had limited long‐term success. In experimental neurobiology, fear extinction is a model for exposure therapy. In this behavioral paradigm, animals are repeatedly exposed in a safe environment to the fearful stimulus, which leads to greatly reduced fear. Studying animal models of extinction already has lead to better therapeutic strategies and development of new candidate drugs. Lack of a powerful genetic model of extinction, however, has limited progress in identifying underlying molecular and genetic factors. In this study, we established a robust behavioral paradigm to study the short‐term effect (acquisition) of extinction in Drosophila melanogaster. We focused on the extinction of olfactory aversive 1‐day memory with a task that has been the main workhorse for genetics of memory in flies. Using this paradigm, we show that extinction can inhibit each of two genetically distinct forms of consolidated memory. We then used a series of single‐gene mutants with known impact on associative learning to examine the effects on extinction. We find that extinction is intact in each of these mutants, suggesting that extinction learning relies on different molecular mechanisms than does Pavlovian learning.