Like extinction, latent inhibition of conditioned fear in mice is blocked by systemic inhibition of L-type voltage-gated calcium channels

Having recently shown that extinction of conditioned fear depends on L-type voltage-gated calcium channels (LVGCCs), we have been seeking other protocols that require this unusual induction mechanism. We tested latent inhibition (LI) of fear, because LI resembles extinction except that cue exposures precede, rather than follow, cue-shock pairing. Systemic injections of two LVGCC inhibitors, nifedipine and diltiazem, before pre-exposure blocked LI completely with no evidence of state-dependent learning. The results indicate that extinction and LI share a common molecular requirement and may support the notion that LI, like extinction, is a form of inhibitory learning.     

The L-type calcium channel blocker nifedipine impairs extinction, but not reduced contingency effects, in mice

We recently reported that fear extinction, a form of inhibitory learning, is selectively blocked by systemic administration of L-type voltage-gated calcium channel (LVGCC) antagonists, including nifedipine, in mice. We here replicate this finding and examine three reduced contingency effects after vehicle or nifedipine (40 mg/kg) administration. In the first experiment, contingency reduction was achieved by adding USs to the training protocol (degraded contingency), a phenomenon thought to be independent of behavioral inhibition. In the second experiment, contingency reduction was achieved by varying the percentage of CS-US pairing, a phenomenon thought to be weakly dependent on behavioral inhibition. In the third and fourth experiments, contingency reduction was achieved by adding CSs to the training protocol (partial reinforcement), a phenomenon thought to be completely dependent on behavioral inhibition. We found that none of these reduced contingency effects was impaired by nifedipine. In a final experiment, we found that extinction conducted 1 or 3 h post-acquisition, but not immediately, was LVGCC-dependent. Taken together, the results suggest that reduced contingency effects and extinction depend on different molecular mechanisms and that LVGCC dependence of behavioral inhibition develops with time after associative CS-US learning.     

The basolateral amygdala is necessary for learning but not relearning extinction of context conditioned fear

Extinction of conditioned fear involves new learning that inhibits but does not eliminate the original fear memory. This inhibitory learning is thought to require activation of NMDA receptors (NMDAr) within the basolateral amygdala (BLA). However, once extinction has been learned, the role played by the BLA during subsequent extinction procedures remains unknown. The present study examined the role of neuronal activity and NMDAr activation in rats receiving their first or second extinction of context fear. We found that BLA infusion of DL-APV, a competitive antagonist of NMDAr, depressed fear responses at both the first and second extinction. It impaired learning extinction but spared and even facilitated relearning extinction. BLA infusion of muscimol, a GABA(A) agonist, produced a similar outcome, suggesting that DL-APV not only blocked NMDAr-dependent plasticity but also disrupted neuronal activity. In contrast, infusion of ifenprodil, a more selective antagonist of NMDAr containing the NR2B subunit, did not depress fear responses but impaired short- and long-term inhibition of fear at both the first and second extinction. Therefore, we suggest that relearning extinction normally requires NMDAr containing the NR2B subunit in the BLA. However, simultaneous blockade of these receptors and neuronal activity in the BLA results in compensatory learning that is able to promote long-term re-extinction. These data are consistent with a current model that attributes fear extinction to interactions between several neural substrates, including the amygdala and the medial prefrontal cortex.     

L-type voltage-gated calcium channels in conditioned fear: a genetic and pharmacological analysis

Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear using a molecular genetics approach. Previously, we used this approach to demonstrate that the Ca(v)1.3 isoform mediates consolidation, but not extinction, of contextually conditioned fear. Here, we used mice in which the gene for the L-VGCC pore-forming subunit Ca(v)1.2 was conditionally deleted in forebrain excitatory neurons (Ca(v)1.2(cKO) mice) to address the role of Ca(v)1.2 in consolidation and extinction of conditioned fear. We demonstrate that Ca(v)1.2(cKO) mice consolidate and extinguish conditioned fear as well as control littermates. These data suggest that Ca(v)1.2 is not critical for these processes and together with our previous data argue against a role for either of the brain-expressed L-VGCCs (Ca(v)1.2 or Ca(v)1.3) in extinction of conditioned fear. Additionally, we present data demonstrating that the L-VGCC antagonist nifedipine, which has been used in previous conditioned fear extinction studies, impairs locomotion, and induces an aversive state. We further demonstrate that this aversive state can enter into associations with conditioned stimuli that are present at the time that it is experienced, suggesting that previous studies using nifedipine were likely confounded by drug toxicity. Taken together, our genetic and pharmacological data argue against a role for Ca(v)1.2 in consolidation of conditioned fear as well as a role for L-VGCCs in extinction of conditioned fear.     

Nonassociative learning processes determine expression and extinction of conditioned fear in mice

Freezing to a tone following auditory fear conditioning is commonly considered as a measure of the strength of the tone-shock association. The decrease in freezing on repeated nonreinforced tone presentation following conditioning, in turn, is attributed to the formation of an inhibitory association between tone and shock that leads to a suppression of the expression of fear. This study challenges these concepts for auditory fear conditioning in mice. We show that acquisition of conditioned fear by a few tone-shock pairings is accompanied by a nonassociative sensitization process. As a consequence, the freezing response of conditioned mice seems to be determined by both associative and nonassociative memory components. Our data suggest that the intensity of freezing as a function of footshock intensity is primarily determined by the nonassociative component, whereas the associative component is more or less categorical. We next demonstrate that the decrease in freezing on repeated nonreinforced tone presentation following conditioning shows fundamental properties of habituation. Thus, it might be regarded as a habituation-like process, which abolishes the influence of sensitization on the freezing response to the tone without affecting the expression of the associative memory component. Taken together, this study merges the dual-process theory of habituation with the concept of classical fear conditioning and demonstrates that sensitization and habituation as two nonassociative learning processes may critically determine the expression of conditioned fear in mice.     

Zinc transporter 3 is involved in learned fear and extinction, but not in innate fear

Synaptically released Zn2+ is a potential modulator of neurotransmission and synaptic plasticity in fear-conditioning pathways. Zinc transporter 3 (ZnT3) knock-out (KO) mice are well suited to test the role of zinc in learned fear, because ZnT3 is colocalized with synaptic zinc, responsible for its transport to synaptic vesicles, highly enriched in the amygdala-associated neural circuitry, and ZnT3 KO mice lack Zn2+ in synaptic vesicles. However, earlier work reported no deficiency in fear memory in ZnT3 KO mice, which is surprising based on the effects of Zn2+ on amygdala synaptic plasticity. We therefore reexamined ZnT3 KO mice in various tasks for learned and innate fear. The mutants were deficient in a weak fear-conditioning protocol using single tone-shock pairing but showed normal memory when a stronger, five-pairing protocol was used. ZnT3 KO mice were deficient in memory when a tone was presented as complex auditory information in a discontinuous fashion. Moreover, ZnT3 KO mice showed abnormality in trace fear conditioning and in fear extinction. By contrast, ZnT3 KO mice had normal anxiety. Thus, ZnT3 is involved in associative fear memory and extinction, but not in innate fear, consistent with the role of synaptic zinc in amygdala synaptic plasticity.     

Role of L-type Ca2+ channel isoforms in the extinction of conditioned fear

Dihydropyridine (DHP) L-type Ca(2+) channel (LTCC) antagonists, such as nifedipine, have been reported to impair the extinction of conditioned fear without interfering with its acquisition. Identification of the LTCC isoforms mediating this DHP effect is an essential basis to reveal their role as potential drug targets for the treatment of specific anxiety disorders. Ca(V)1.2 and Ca(V)1.3 are the predominant LTCCs in the mammalian brain. However, since no isoform-selective DHP blockers are available, their individual contribution to fear memory extinction is unknown. We used a novel mouse model expressing DHP-insensitive Ca(V)1.2 LTCCs (Ca(V)1.2DHP(-/-) mice) to address this question. In line with previous studies, wild-type (WT) mice treated with systemic nifedipine displayed markedly impaired fear extinction. This DHP effect was completely abolished in Ca(V)1.2DHP(-/-) mice, indicating that it is mediated by Ca(V)1.2, but not by Ca(V)1.3 LTCCs. Supporting this conclusion, Ca(V)1.3-deficient mice (Ca(V)1.3(-/-)) showed extinction identical to the respective WT mice. The inhibition of fear extinction was not observed after intracerebroventricular (i.c.v.) application of different doses of nifedipine, suggesting that this effect is secondary to inhibition of peripheral Ca(V)1.2 channels. The LTCC activator BayK, which lacks neurotoxic effects in Ca(V)1.2DHP(-/-) mice, did not influence the extinction time course. In summary, we demonstrate that LTCC signaling through the Ca(V)1.2 isoform of LTCCs interferes with fear memory extinction, presumably via a peripherally mediated mechanism. Activation of other LTCC isoforms (predominantly Ca(V)1.3) is not sufficient to accelerate extinction of conditioned fear in mice.     

The effect of hypocapnia on extinction of conditioned fear responses

Conditioning models have been very helpful in the understanding of the etiology and maintenance of anxiety. Such laboratory models, however, leave unexplained why in many cases of naturally occurring anxiety, as in the case of agoraphobia, the fear responses do not extinguish. Literature on experimental anxiety provocation suggests that a systemic alkalosis might play a role in the maintenance of phobic fear. It was hypothesized that a subject in a state of respiratory alkalosis would show delayed extinction to classical conditioned anxiety. In a differential classical conditioning paradigm, consisting of a habituation-, an acquisition-, and an extinction-phase, slides and electric shocks were used as conditioned stimuli (CS) and unconditioned stimuli (US) respectively. The skin conductance response was taken as (U)CR. Subjects were randomly assigned to two groups: hyperventilation or control. It was shown that the extinction was not delayed when subjects were hypocapnic during the extinction. These data support the view that a respiratory alkalosis per se is not a sufficient condition for the maintenance of neurotic fears. The data of the present study are discussed in the context of existing literature on a psychological interpretation of the maintenance of anxiety.     

Systemic blockade of D2-like dopamine receptors facilitates extinction of conditioned fear in mice

Extinction of conditioned fear in animals is the explicit model of behavior therapy for human anxiety disorders, including panic disorder, obsessive-compulsive disorder, and post-traumatic stress disorder. Based on previous data indicating that fear extinction in rats is blocked by quinpirole, an agonist of dopamine D2 receptors, we hypothesized that blockade of D2 receptors might facilitate extinction in mice, while agonists should block extinction, as they do in rats. One day after fear conditioning mice with three pairings of a white noise conditional stimulus (CS) with moderate footshock, we injected the D2 antagonist, sulpiride, the D2 agonist, quinpirole, or vehicle, just before repeated CS presentations to generate extinction. We assayed fear by measuring behavioral freezing during extinction presentations and then drug-free during CS presentations 1 d later. We found that sulpiride injections before extinction training facilitated extinction memory 24 h later, while quinpirole partially blocked extinction memory compared with vehicle-injected controls. Notably, sulpiride treatment yielded significant extinction after spaced CS presentations, which yield no extinction at all in vehicle-treated mice. These findings suggest that dopamine D2-mediated signaling contributes physiological inhibition of extinction, and that D2 antagonists may be useful adjuncts to behavior therapy of human anxiety disorders.     

提取-消退范式中复合刺激对恐惧消退的影响

情绪障碍治疗的关键在于消退条件性恐惧记忆,研究证明基于记忆再巩固的提取-消退范式能有效消除或改写原有的恐惧记忆。本研究将提取-消退范式应用到更复杂的恐惧记忆中,采用多感官复合刺激(声音+图片)作为条件刺激,以皮电反应作为恐惧反应指标,考察采用单个线索(声音或图片)、复合线索(声音+图片)进行提取-消退对条件性恐惧记忆的消退效果有何差异。结果表明:声音线索提取-消退组出现了自发恢复和重建效应,图片提取-消退组只出现了重建效应,复合刺激提取-消退组未出现自发恢复和重建效应。说明由复合刺激线索引发的条件性恐惧,采用复合刺激中的单个较强线索或原有完整线索进行提取-消退,对恐惧记忆的消退效果最好。     

Concurrent excitors limit the extinction of conditioned fear in humans

In a human fear conditioning experiment, with on-line expectancy ratings and electrodermal responding as indices of fear, two neutral stimuli (pictures of geometric shapes) were first established as reliable predictors of an electric shock. In the subsequent extinction phase, the two stimuli were repeatedly presented in compound, without the shock. The final test phase consisted of individual stimulus presentations again, which resulted in a strong return of the conditioned responses. This effect was not observed in non-conditioned control stimuli. Hence, behavioral effects of extinction seem highly specific to the stimulus constellation that has gone through the extinction procedure. We argue that pharmacological, behavioral and/or cognitive manipulations that could prevent configural processing of stimulus constellations have direct clinical potential.     

Microinfusion of the D1 receptor antagonist, SCH23390 into the IL but not the BLA impairs consolidation of extinction of auditory fear conditioning

In auditory fear conditioning, repeated presentation of the tone in the absence of the shock leads to extinction of the acquired fear response. Both the medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) are involved in extinction. Here we examined this involvement by antagonizing D1 receptors in both regions, in the rat. We microinfused the D1 receptor antagonist, SCH23390, into the infra-limbic part of the mPFC (IL) or BLA at different time points. SCH23390 mircoinfused into the IL either before extinction acquisition or following short extinction training resulted in impairment of extinction consolidation. Microinfusion of SCH23390 into the BLA, prior to acquisition of extinction caused impairment in acquisition of extinction without affecting extinction consolidation. This is supported by the results showing that microinfusion of SCH23390 into the BLA following a short-training session did not affect consolidation. These results further strengthen the role of mPFC in consolidation of extinction while highlighting the role of the D1 receptors in this process.     

CB1 cannabinoid receptors modulate kinase and phosphatase activity during extinction of conditioned fear in mice

Cannabinoid receptors type 1 (CB1) play a central role in both short-term and long-term extinction of auditory-cued fear memory. The molecular mechanisms underlying this function remain to be clarified. Several studies indicated extracellular signal-regulated kinases (ERKs), the phosphatidylinositol 3-kinase with its downstream effector AKT, and the phosphatase calcineurin as potential molecular substrates of extinction behavior. To test the involvement of these kinase and phosphatase activities in CB1-dependent extinction of conditioned fear behavior, conditioned CB1-deficient mice (CB1(-/-)) and wild-type littermates (CB1(+/+)) were sacrificed 30 min after recall of fear memory, and activation of ERKs, AKT, and calcineurin was examined by Western blot analysis in different brain regions. As compared with CB1(+/+), the nonreinforced tone presentation 24 h after auditory-cued fear conditioning led to lower levels of phosphorylated ERKs and/or calcineurin in the basolateral amygdala complex, ventromedial prefrontal cortex, dorsal hippocampus, and ventral hippocampus of CB1(-/-). In contrast, higher levels of phosphorylated p44 ERK and calcineurin were observed in the central nucleus of the amygdala of CB1(-/-). Phosphorylation of AKT was more pronounced in the basolateral amygdala complex and the dorsal hippocampus of CB1(-/-). We propose that the endogenous cannabinoid system modulates extinction of aversive memories, at least in part via regulation of the activity of kinases and phosphatases in a brain structure-dependent manner.     

The histone deacetylase inhibitor valproic acid enhances acquisition, extinction, and reconsolidation of conditioned fear

Histone modifications contribute to the epigenetic regulation of gene expression, a process now recognized to be important for the consolidation of long-term memory. Valproic acid (VPA), used for many years as an anticonvulsant and a mood stabilizer, has effects on learning and memory and enhances the extinction of conditioned fear through its function as a histone deacetylase inhibitor (HDAC). Here we report that VPA enhances long-term memory for both acquisition and extinction of cued-fear. Interestingly, VPA enhances extinction, but also enhances renewal of the original conditioned fear when tested in a within-subjects design. This effect appears to be related to a reconsolidation-like process since a single CS reminder in the presence of VPA can enhance long-term memory for the original fear in the context in which fear conditioning takes place. We also show that by modifying the intertrial interval during extinction training, VPA can strengthen reconsolidation of the original fear memory or enhance long-term memory for extinction such that it becomes independent of context. These findings have important implications for the use of HDAC inhibitors as adjuncts to behavior therapy in the treatment of phobia and related anxiety disorders.     

Learning and memory in conditioned fear extinction: effects of D-cycloserine

This review addresses the effects of the cognitive enhancer D-cycloserine (DCS) on the memory processes that occur in conditioned fear extinction, which is the experimental model for exposure techniques to reduce clinical anxiety. All reported rat studies show an enhanced fear extinction effect when DCS is administered acutely before or shortly after extinction training. DCS also promotes the generalization of this fear extinction effect. In addition, DCS reduces some forms of relapse (reduced reinstatement, reduced spontaneous recovery), but not others (contextual renewal, rapid reacquisition). It is argued that this pattern of results is best explained by assuming that DCS promotes extinction learning to the background context, resulting in enhanced contextual inhibition. Four human studies have produced mixed results, but some methodological issues complicate the reported failures. It is concluded that DCS is a promising tool as an adjunct to extinction techniques in exposure treatment, but that more pre-clinical and clinical research is needed to fully characterize its behavioral consequences.     

Inhibition of matrix metalloproteinase activity disrupts reconsolidation but not consolidation of a fear memory

The reconsolidation hypothesis posits that memories that have been reactivated can be either enhanced or disrupted by pharmacological manipulation. Synaptic plasticity is presumed to underlie the reconsolidation process. Matrix metalloproteinases are proteins that regulate the extracellular matrix involved in plasticity events, and these proteins have recently been shown to influence learning and memory. However, all studies on the role of matrix metalloproteinases in learning and memory have employed tasks that rely on contextual cues. The goal of this study was to determine the extent to which FN-439 would disrupt the consolidation and/or reconsolidation of a fear memory associated with a conditioned stimulus that signaled tone-shock pairings and that was independent of contextual cues. Male Sprague-Dawley rats were given infusions of FN-439 (35 microg intracerebroventricular) 30 min prior to conditioning (tone-shock paired association) or 30 min prior to a single reactivation session given 24h after conditioning. Administration of FN-439 did not disrupt consolidation of the freezing response when the tone (conditioned stimulus) was presented. In contrast, FN-439 infusion disrupted reconsolidation of the fear memory in a reactivation-dependent manner. The reduced freezing behavior was not due to a decrease in general anxiety levels, since FN-439 had no effect on the percent of open-arm time or open-arm entries in an elevated-plus maze task. Thus, we demonstrated for the first time that matrix metalloproteinase inhibition in the brain is capable of disrupting the reconsolidation of a tone-shock association memory that does not depend on contextual cues. The finding that a fear response to a previously paired conditioned stimulus can be disrupted by treatment with an MMP inhibitor during a single reactivation session suggests that this class of compounds may have therapeutic potential for posttraumatic stress disorder and/or simple phobias.     

Differential acquisition,extinction, and reinstatement of conditioned suppression in mice

In animals, the reappearance of conditioned fear responses after extinction has been primarily investigated using single-cue conditioning paradigms. However, a differential paradigm can overcome several of the disadvantages associated with a single-cue procedure. In the present study, the reinstatement phenomenon was assessed in mice using a differential conditioned suppression paradigm. In a first phase, one conditioned stimulus (CS + ) was consistently paired with an unconditioned stimulus (US; footshock) while another CS (CS–) was not, resulting in selective suppression of previously trained instrumental behaviour during the CS + . After the extinction phase, half of the animals (reinstatement group) were presented with unsignalled USs, while the other half were not (control group). A differential return of conditioned responding was observed in the reinstatement group, but not in the control group. The implications of these findings for future conditioning research are discussed.     

Differential acquisition, extinction, and reinstatement of conditioned suppression in mice

In animals, the reappearance of conditioned fear responses after extinction has been primarily investigated using single-cue conditioning paradigms. However, a differential paradigm can overcome several of the disadvantages associated with a single-cue procedure. In the present study, the reinstatement phenomenon was assessed in mice using a differential conditioned suppression paradigm. In a first phase, one conditioned stimulus (CS + ) was consistently paired with an unconditioned stimulus (US; footshock) while another CS (CS-) was not, resulting in selective suppression of previously trained instrumental behaviour during the CS + . After the extinction phase, half of the animals (reinstatement group) were presented with unsignalled USs, while the other half were not (control group). A differential return of conditioned responding was observed in the reinstatement group, but not in the control group. The implications of these findings for future conditioning research are discussed.