Amygdala upregulation of NCAM polysialylation induced by auditory fear conditioning is not required for memory formation, but plays a role in fear extinction

There is much interest to understand the mechanisms leading to the establishment, maintenance, and extinction of fear memories. The amygdala has been critically involved in the processing of fear memories and a number of molecular changes have been implicated in this brain region in relation to fear learning. Although neural cell adhesion molecules (NCAMs) have been hypothesized to play a role, information available about their contribution to fear memories is scarce. We investigate here whether polysialylated NCAM (PSA-NCAM) contributes to auditory fear conditioning in the amygdala. First, PSA-NCAM expression was evaluated in different amygdala nuclei after auditory fear conditioning at two different shock intensities. Results showed that PSA-NCAM expression was increased 24 h post-training only in animals subjected to the highest shock intensity (1mA). Second, PSA-NCAM was cleaved in the basolateral amygdaloid complex through micro-infusions of the enzyme endoneuraminidase N, and the consequences of such treatment were investigated on the acquisition, consolidation, remote memory expression, and extinction of conditioned fear memories. Intra-amygdaloid cleavage of PSA-NCAM did not affect acquisition, consolidation or expression of remote fear memories. However, intra-amygdaloid PSA-NCAM cleavage enhanced fear extinction processes. These results suggest that upregulation of PSA-NCAM is a correlate of fear conditioning that is not necessary for the establishment of fear memory in the amygdala, but participates in mechanisms precluding fear extinction. These findings point out PSA-NCAM as a potential target for the treatment of psychopathologies that involve impairment in fear extinction.     

Calcitonin gene-related peptide released within the amygdala is involved in Pavlovian auditory fear conditioning

The effects of CGRP and the CGRP receptor antagonist hCGRP(8-37) injected into the amygdala on both the acquisition and expression of fear behavior to a discrete auditory conditional stimulus (CS) and the training context were assessed. In Experiment 1, pretraining injections of CGRP but not hCGRP(8-37) produced fear-like behavior before any aversive stimuli were presented. While both compounds attenuated freezing to the contextual CS on the test day, neither affected learning about the auditory CS. In Experiment 2, pretesting injections of hCGRP(8-37) (0.63 mM) selectively attenuated freezing to the auditory CS but left freezing to the contextual CS intact. These data suggest that CGRP in the amygdala may selectively contribute to the expression of learning about auditory stimuli during fear conditioning.     

Contextual fear conditioning predicts subsequent avoidance behaviour in a virtual reality environment

Avoidance behaviour is a crucial component of fear and is importantly involved in the maintenance of anxiety disorders. Presumably, fear conditioning leads to avoidance of the feared object or context. A virtual reality contextual fear conditioning paradigm was used to investigate the association between explicit conditioning effects and subsequent avoidance behaviour. Mild electric shocks were administered in one context (anxiety context), but never in a second context (safety context). Subsequent avoidance behaviour was assessed by asking participants to choose two out of three contexts (a neutral context was added) to visit again. Participants avoided the anxiety context, but did not prefer the safety over the neutral context. Participants with substantial conditioning effects, as reflected in differential valence, arousal and anxiety ratings, avoided the anxiety context but not the safety context. In sum, we demonstrated an association between context conditioning effects on an explicit level and later avoidance behaviour.     

Contextual fear conditioning predicts subsequent avoidance behaviour in a virtual reality environment

Avoidance behaviour is a crucial component of fear and is importantly involved in the maintenance of anxiety disorders. Presumably, fear conditioning leads to avoidance of the feared object or context. A virtual reality contextual fear conditioning paradigm was used to investigate the association between explicit conditioning effects and subsequent avoidance behaviour. Mild electric shocks were administered in one context (anxiety context), but never in a second context (safety context). Subsequent avoidance behaviour was assessed by asking participants to choose two out of three contexts (a neutral context was added) to visit again. Participants avoided the anxiety context, but did not prefer the safety over the neutral context. Participants with substantial conditioning effects, as reflected in differential valence, arousal and anxiety ratings, avoided the anxiety context but not the safety context. In sum, we demonstrated an association between context conditioning effects on an explicit level and later avoidance behaviour.     

Prolactin in rats is attenuated by conspecific touch in a novel environment

Prolactin is released in response to physical and psychological stress, the latter of which involves an animal’s interpretation of its environment as potentially dangerous. This interpretation might be altered by social buffering, as defined by the presence of a social partner. In support of this claim, I previously found that the presence of a conspecific during exposure to the open field caused lower levels of prolactin in juvenile rats than did exposure to the open field alone. Because testing with a conspecific allowed the animals to touch, physical contact may have been necessary for the reduction of prolactin. To test this hypothesis, juvenile male and female rats in the present study were exposed to the open field (1) alone, (2) with a conspecific separated by a Plexiglas partition, or (3) with a conspecific without a partition. Touch was necessary to reduce stress as measured by prolactin levels.     

Return of fear in a human differential conditioning paradigm caused by a stimulus change after extinction

In a human fear conditioning experiment, 32 participants were trained in a differential conditioning procedure with geometrical shapes as CS+ and CS- (four presentations each), and an electric shock as US. Measures of conditioned responding were skin conductance response (SCR) and retrospective US-expectancy ratings. For half of the participants (Generalization Group, GG), the subsequent extinction phase consisted of four nonreinforced presentations of generalization stimuli (GS+ and GS-). Participants from the Extinction control Group received an equal amount of nonreinforced presentations of the CSs. Finally, all participants were tested with the original CSs. The results from both measures clearly show an increase in the size of the discrimination upon the stimulus change after extinction in the GG. Because this pattern is not observed in the Extinction control Group, extinction performance appears to be somehow restricted to the perceptual characteristics of the extinction stimulus. Interestingly, the size of the conditioned SCR discrimination in the GG is not influenced by the stimulus change after acquisition. This observation points to a differential impact of stimulus change after acquisition vs. extinction treatment. The findings are discussed from the theoretical perspective of renewal and the clinical perspective of Return of Fear.     

Contextual,but not auditory,fear conditioning is disrupted by neurotoxic selective lesion of the basal nucleus of amygdala in rats

The basolateral amygdala complex (BLA) is involved in acquisition of contextual and auditory fear conditioning. However, the BLA is not a single structure but comprises a group of nuclei, including the lateral (LA), basal (BA) and accessory basal (AB) nuclei. While it is consensual that the LA is critical for auditory fear conditioning, there is controversy on the participation of the BA in fear conditioning. Hodological and neurophysiological findings suggest that each of these nuclei processes distinct information in parallel; the BA would deal with polymodal or contextual representations, and the LA would process unimodal or elemental representations. Thus, it seems plausible to hypothesize that the BA is required for contextual, but not auditory, fear conditioning. This hypothesis was evaluated in Wistar rats submitted to multiple-site ibotenate-induced damage restricted to the BA and then exposed to a concurrent contextual and auditory fear conditioning training followed by separated contextual and auditory conditioning testing. Differing from electrolytic lesion and lidocaine inactivation, this surgical approach does not disturb fibers of passage originating in other brain areas, restricting damage to the aimed nucleus. Relative to the sham-operated controls, rats with selective damage to the BA exhibited disruption of performance in the contextual, but not the auditory, component of the task. Thus, while the BA seems required for contextual fear conditioning, it is not critical for both an auditory-US association, nor for the expression of the freezing response.     

The amygdala is not necessary for unconditioned stimulus inflation after Pavlovian fear conditioning in rats

The basolateral complex (BLA) and central nucleus (CEA) of the amygdala play critical roles in associative learning, including Pavlovian conditioning. However, the precise role for these structures in Pavlovian conditioning is not clear. Recent work in appetitive conditioning paradigms suggests that the amygdala, particularly the BLA, has an important role in representing the value of the unconditioned stimulus (US). It is not known whether the amygdala performs such a function in aversive paradigms, such as Pavlovian fear conditioning in rats. To address this issue, Experiments 1 and 2 used temporary pharmacological inactivation of the amygdala prior to a US inflation procedure to assess its role in revaluing shock USs after either overtraining (Experiment 1) or limited training (Experiment 2), respectively. Inactivation of the BLA or CEA during the inflation session did not affect subsequent increases in conditioned freezing observed to either the tone conditioned stimulus (CS) or the conditioning context in either experiment. In Experiment 3, NBQX infusions into the BLA impaired the acquisition of auditory fear conditioning with an inflation-magnitude US, indicating that the amygdala is required for associative learning with intense USs. Together, these results suggest that the amygdala is not required for revaluing an aversive US despite being required for the acquisition of fear to that US.Pavlovian fear conditioning in rats is a behavioral model used to investigate the neurobiology underlying the development and maintenance of fear learning and memory (Grillon et al. 1996; LeDoux 1998, 2000; Bouton et al. 2001; Maren 2001b, 2005; Kim and Jung 2006). In this model, an innocuous conditioned stimulus (CS), such as a tone, is paired with an aversive unconditioned stimulus (US), such as a footshock. After one or more pairings, the rat learns that the CS predicts the US. As a consequence, CS presentations alone elicit a conditioned fear response (CR), which includes increases in heart rate, arterial blood pressure, hypoalgesia, potentiated acoustic startle, stress hormone release, and freezing (somatomotor immobility).The amygdala has been identified as one of the major regions in which fear memories are encoded and stored. Within the amygdala, the basolateral complex of the amygdala (BLA; consisting of the lateral, basolateral, and basomedial nuclei) and the central nucleus of the amygdala (CEA) receive convergent CS and US information and are involved in the acquisition of fear memories (LeDoux 1998, 2000; Fendt and Fanselow 1999; Davis and Whalen 2001; Maren 2001b; Schafe et al. 2001; Fanselow and Gale 2003; Wilensky et al. 2006; Zimmerman et al. 2007). In addition, the CEA has an important role in the expression of fear CRs (Fendt and Fanselow 1999; LeDoux 2000; Davis and Whalen 2001; Maren 2001b; Fanselow and Gale 2003). In support of this, many studies have shown that either permanent or temporary lesions of the BLA or CEA prevent the acquisition and/or expression of fear memories (Helmstetter 1992; Helmstetter and Bellgowan 1994; Campeau and Davis 1995; Maren et al. 1996a,b; Killcross et al. 1997; Muller et al. 1997; Walker and Davis 1997; Cousens and Otto 1998; Maren 1998, 1999, 2001a,b; Wilensky et al. 1999, 2000, 2006; Goosens and Maren 2001, 2003; Nader et al. 2001; Fanselow and Gale 2003; Gale et al. 2004; Koo et al. 2004; Zimmerman et al. 2007).In addition to its role in encoding CS–US associations during conditioning, recent work suggests that the amygdala is also involved in representing properties of the US itself. For example, temporary or permanent lesions of the BLA reduce both decrements in conditioned responding after devaluation of a food US (Hatfield et al. 1996; Killcross et al. 1997; Blundell et al. 2001; Balleine et al. 2003; Everitt et al. 2003; Pickens et al. 2003; Holland 2004) and increments in conditional responding after inflation of a shock US (Fanselow and Gale 2003). Moreover, recent electrophysiological studies in primates indicate that amygdala neurons represent the value of both aversive and appetitive outcomes (Paton et al. 2006; Belova et al. 2007, 2008; Salzman et al. 2007). These studies suggest that one function of the BLA is to represent specific properties of biologically significant events, such as the food or shock USs that are typically used in Pavlovian conditioning paradigms. By this view, the BLA may represent specific sensory properties of USs that shape the nature of learned behavioral responses to the US (Balleine and Killcross 2006) and allow CSs to gain access to the incentive value of the US (Everitt et al. 2003).In contrast to this view, we recently reported that rats with neurotoxic BLA lesions exhibit normal US revaluation after Pavlovian fear conditioning (Rabinak and Maren 2008). In this study, auditory fear conditioning (75 CS–US trials) with a moderate footshock (1 mA) was followed by several exposures (five US-alone trials) to an intense footshock (3 mA) during an inflation session. Both intact rats and rats with BLA lesions exhibit a robust increase in conditional freezing to the auditory CS during a subsequent retention test (Rabinak and Maren 2008). Control experiments suggested that this was due to a revaluation of the US with which the CS was associated, rather than nonassociative sensitization of fear engendered by exposure to intense shock. These data reveal that the BLA may not be necessary for representing properties of shock USs during Pavlovian fear conditioning. To address these issues further, we have examined the consequence of reversible pharmacological manipulations of the amygdala during US inflation on conditional fear responses established with either extensive or limited training.     

Enduring deficits in contextual and auditory fear conditioning after adolescent, not adult, social instability stress in male rats

Adolescence is a time of developmental changes and reorganization in the brain and stress systems, thus, adolescents may be more vulnerable than adults to the effects of chronic mild stressors. Most studies, however, have not directly compared stress experienced in adolescence to the same stress experience in adulthood. In the present study, adolescent (n=46) and adult (n=48) male rats underwent 16 days of social instability stress (daily 1h isolation and change of cage partners) or were non-stress controls. Rats were then tested on the strength of acquired contextual and cued fear conditioning, as well as extinction learning, beginning either the day after the stress procedure or 3 weeks later. No difference was found among the groups during the Training Phase of conditioning. Irrespective of the time between the social stress experience and fear conditioning, rats stressed in adolescence had decreased context and cue memory, and cue generalization compared to control rats, as measured by the percentage of time spent freezing in tests. Social instability stress in adulthood had no effect on any measure of fear conditioning. The results support the hypothesis that adolescence is a time of heightened vulnerability to stressors.     

Contextual and auditory fear conditioning are mediated by the lateral, basal, and central amygdaloid nuclei in rats

A large body of literature implicates the amygdala in Pavlovian fear conditioning. In this study, we examined the contribution of individual amygdaloid nuclei to contextual and auditory fear conditioning in rats. Prior to fear conditioning, rats received a large electrolytic lesion of the amygdala in one hemisphere, and a nucleus-specific neurotoxic lesion in the contralateral hemisphere. Neurotoxic lesions targeted either the lateral nucleus (LA), basolateral and basomedial nuclei (basal nuclei), or central nucleus (CE) of the amygdala. LA and CE lesions attenuated freezing to both contextual and auditory conditional stimuli (CSs). Lesions of the basal nuclei produced deficits in contextual and auditory fear conditioning only when the damage extended into the anterior divisions of the basal nuclei; damage limited to the posterior divisions of the basal nuclei did not significantly impair conditioning to either auditory or contextual CS. These effects were typically not lateralized, although neurotoxic lesions of the posterior divisions of the basal nuclei had greater effects on contextual fear conditioning when the contralateral electrolytic lesion was placed in the right hemisphere. These results indicate that there is significant overlap within the amygdala in the neural pathways mediating fear conditioning to contextual and acoustic CS, and that these forms of learning are not anatomically dissociable at the level of amygdaloid nuclei.     

Origins of fear of dogs in adults and children: the role of conditioning processes and prior familiarity with dogs.

One hundred adults and 30 children completed questionnaires to investigate fear of dogs. Dog fearful adults asked to recall the origins of their fear reported classical conditioning experiences more frequently than vicarious acquisition or informational transmission. Overall, however, there was no difference in the frequency of attacks reported by the fearful and non-fearful groups. Significantly more fearful than non-fearful adults reported little contact with dogs prior to the onset of their fear which suggests that early non-eventful exposure to dogs may prevent a conditioning event from producing a dog phobia. Most adults reported that their fear began in childhood, and dog fear were more frequently reported by children than by adults. In the aggregate, however, dog-fearful adults and children differed in several ways; children were more likely than adults to report having received warnings about dogs, but also to recognize the potential attractiveness of a friendly dog. Unlike dog-fearful children, dog-fearful adults reported many other fears in addition to their fear of dogs. A better understanding of fear of dogs in adults may depend on discovering why some dog-fearful children, but not others, apparently lose their fear of dogs as they become older.     

Early extinction after fear conditioning yields a context-independent and short-term suppression of conditional freezing in rats

Extinction of Pavlovian fear conditioning in rats is a useful model for therapeutic interventions in humans with anxiety disorders. Recently, we found that delivering extinction trials soon (15 min) after fear conditioning yields a short-term suppression of fear, but little long-term extinction. Here, we explored the possible mechanisms underlying this deficit by assessing the suppression of fear to a CS immediately after extinction training (Experiment 1) and the context specificity of fear after both immediate and delayed extinction training (Experiment 2). We also examined the time course of the immediate extinction deficit (Experiment 3). Our results indicate that immediate extinction produces a short-lived and context-independent suppression of conditional freezing. Deficits in long-term extinction were apparent even when the extinction trials were given up to 6 h after conditioning. Moreover, this deficit was not due to different retention intervals that might have influenced the degree of spontaneous recovery after immediate and delayed extinction (Experiment 4). These results suggest that fear suppression under immediate extinction may be due to a short-term, context-independent habituation process, rather than extinction per se. Long-term extinction memory only develops when extinction training occurs at least six hours after conditioning.Pavlovian fear conditioning and extinction are important behavioral models for studying the brain mechanisms underlying the acquisition, storage, retrieval, and suppression of traumatic fear (LeDoux 2000; Maren 2001, 2005; Kim and Jung 2005). In this procedure, an emotionally neutral stimulus, such as a tone, is paired with an aversive stimulus (US), such as an electric foot shock. After a few tone–foot shock pairings, the previous neutral tone becomes a potent conditioned stimulus (CS) and acquires the ability to elicit fear responses, such as freezing (CR). However, with repeated presentations of the CS-alone, the previously acquired CR gradually subsides, a process called extinction (Davis et al. 2003; Maren and Quirk 2004; Kim and Jung 2005; Myers and Davis 2007). The behavioral processes and the underlying neural mechanisms of extinction have attracted extensive attention in contemporary research of learning and memory (Bouton et al. 2006). Indeed, it has been suggested that failure to extinguish fear may contribute to post-traumatic stress disorder (PTSD) (Bouton et al. 2001; Rothbaum and Davis 2003). To avoid the possible long-term consequences and costs of PTSD or other anxiety disorders, clinical interventions are essential. While early interventions may manage the stress response to trauma, their efficacy has been challenged, because the acute intense stress of the traumatic experience might actually exacerbate relapse of fear (McNally 2003; Rothbaum and Davis 2003; Gray and Litz 2005). Thus, it is essential to learn when these interventions generate the best long-term extinction of fear responses.In a recent study, we demonstrated that delivering extinction trials shortly after fear conditioning yields poor long-term fear reduction (Maren and Chang 2006; but, see Myers et al. 2006). We observed that conditional freezing decreased during extinction training, but recovered completely 24 h later. This was true even when we gave 225 massed extinction trials 15 min after fear conditioning. However, in these experiments the within-session decrease in fear in rats that underwent extinction was similar to that in rats that were not exposed to extinction trials. Thus, it is unclear to what extent the short-term fear suppression we observed was due to a loss of fear to the context, the auditory CS, or both. It is also not clear whether fear suppression was due to extinction or, alternatively, another learning process such as habituation.To examine these issues further, in the present study we first assessed fear suppression to the auditory CS after immediate extinction by probing CS fear 15 min after extinction training. In a second experiment, we examined whether short-term fear suppression to the CS is renewed outside of the extinction context, as context specificity is one of the hallmarks of extinction (Bouton 2002; Ji and Maren 2007). In the third and fourth experiments, we examined the temporal delay necessary between conditioning and extinction to yield long-term suppression of fear. In our previous work (Maren and Chang 2006), all phases of training were conducted in the same context. Therefore, fear to the context decreased conditional freezing to the tone, particularly when extinction occurred shortly after conditioning, a time at which sensitized context fear was high. In an effort to isolate fear to the tone CS during extinction, we conducted extinction and test sessions in a context that was different from the conditioning context (i.e., an ABB procedure, where each letter denotes the context used for conditioning, extinction, and test, respectively). Our results reveal that delivering CS-alone trials shortly after fear conditioning produces a short-lived and context-independent suppression of freezing. This fear suppression may be due to a short-term, context-independent habituation process, rather than extinction. Furthermore, poor long-term extinction occurs even when the extinction trials were administered up to 6 h after conditioning.     

The decrement in conditioned fear with increased trials of simultaneous conditioning is not specific to the simultaneous procedure

Three experiments using a conditioned punishment paradigm with rat subjects examined the possibility that the nonmonotonic acquisition function previously found to characterize simultaneous conditioning was due to the noninformative nature of the conditioned stimulus (CS). In Experiment 1 the suppressive effects of a CS previously presented with an unconditioned stimulus (US) in a simultaneous and forward (informative) manner were compared following 20 and an additional 60 conditioning trials. Excitatory conditioning similarly diminished with increased trials for both the simultaneous and forward procedures. Experiment 2 employed a between-groups design. Simultaneous, forward, and trace conditioning procedures were compared following 20 or 100 trials. Each of the three 100-trial groups showed less resistance to extinction than their 20-trial counterparts. Experiment 3 determined that the decrement in excitatory conditioning for the 100-trial groups was not due to the greater number of US presentations, per se, but rather to the number of CS-US pairings. The nonmonotonic acquisition function observed with all three conditioning procedures indicated that informational factors were not responsible for the decrement observed in simultaneous conditioning. The pattern of results suggested that subjects receiving extended conditioning trials were better able to discriminate between training and testing.     

Trace fear conditioning is enhanced in mice lacking the delta subunit of the GABAA receptor

The delta subunit of the GABA(A) receptor (GABA(A)R) is highly expressed in the dentate gyrus of the hippocampus. Genetic deletion of this subunit reduces synaptic and extrasynaptic inhibition and decreases sensitivity to neurosteroids. This paper examines the effect of these changes on hippocampus-dependent trace fear conditioning. Compared to controls, delta knockout mice exhibited enhanced acquisition of tone and context fear. Hippocampus-independent delay conditioning was normal in these animals. These results suggest that reduced inhibition in the dentate gyrus facilitates the acquisition of trace fear conditioning. However, the enhancement in trace conditioning was only observed in female knockout mice. The sex-specificity of this effect may be a result of neuroactive steroids. These compounds vary during the estrus cycle, can increase GABAergic inhibition, and have been shown to impair hippocampus-dependent learning. We propose that activation of GABA(A)Rs by neuroactive steroids inhibits learning processes in the hippocampus. Knockouts are immune to this effect because of the reduced neurosteroid sensitivity that accompanies deletion of the delta subunit. Relationships between neurosteroids, hippocampal excitability, and memory are discussed.     

The development of cued versus contextual conditioning in a predictable and an unpredictable human fear conditioning preparation

In this human fear conditioning study, the online development of conditioned US-expectancy to discrete cues and background contexts was measured in two groups. In the paired group (n=30), the CS was systematically followed by an aversive shock (US). In the unpaired group (n=30), CS and US were presented explicitly unpaired. Using US-expectancy ratings, we replicated the basic finding already illustrated in humans with startle modulation. In the paired group, the CS elicited more US-expectancy than the context, whereas in the unpaired group, the context elicited more US-expectancy than the CS. Interestingly, we also observed a trial-by-trial development of conditioning to the context in the unpaired group as indicated by a significant linear trend. This gradual development and the evidence for the role of US-expectancy in contextual fear add to the idea that cued and contextual fear rely on the same basic associative processes.