A modified counterconditioning procedure prevents the renewal of conditioned fear in rats

Two studies using an ABA design examined the Extinction and renewal of conditioned barpress suppression. Following lights-off and foot shock pairings in Context A, rats were placed in Context B and were given either a standard counterconditioning procedure where the lights-off CS was paired with a novel food US delivered freely or a modified counterconditioning procedure where CS-US pairings only occurred if the rat earned the US by performing a required behavior during the CS. Results indicated that the modified counterconditioning procedure thwarted ABA renewal but the conventional counterconditioning procedure did not reduce ABA renewal any more than nonreinforced exposure to the CS alone. Furthermore, the response required during the modified counterconditioning procedure could be one used as a baseline response during Acquisition of fear or it could be a novel response. Implications of the results for theories of Extinction and renewal of fear are discussed.     

Role of context similarity in ABA, ABC, and AAB renewal paradigms: Implications for theories of renewal and for treating human phobias

Using barpress conditioned suppression, we studied the renewal of conditioned fear in rats, an animal model for the relapse of human fears and phobias. We demonstrated ABA renewal when the only differences between Contexts A and B included (1) their odor, (2) their location (i.e., side of room), and (3) unintended differences between copies of the same box at the two sites. Removing either the odor or location cues abolished the renewal effect. We then directly compared the effects of ABA and AAB procedures under two levels of context similarity. Although AAB renewal occurred, ABA renewal was stronger. Adding multiple context distinctions to the three listed above did not significantly enhance either form of renewal. Finally, we directly compared the strengths of AAB, ABC, and ABA renewal. AAB renewal, though again significant, was weaker than ABA and ABC renewal, which did not differ significantly. Fear renewal (relapse) can thus be reduced by extinguishing the fear in the acquisition context, regardless of the nature of the test context.     

Extensive extinction in multiple contexts eliminates the renewal of conditioned fear in rats

Two studies examined whether nonreinforcement of a stimulus in multiple contexts, instead of a single context, would decrease renewal of conditioned fear in rats (as assessed by conditioned suppression of lever pressing). In Experiment 1, renewal was measured after 36 nonreinforced CS trials delivered during six extinction sessions in a single context or two extinction sessions in each of three different contexts. The number of extinction contexts did not have an effect on renewal. In Experiment 2, groups received either 36 or 144 nonreinforced CS trials during six or twenty-four extinction sessions in a single context or three different contexts. Again, renewal was not influenced by the number of extinction contexts when only 36 trials were given. However, when 144 trials were used, renewal was completely eliminated when extinction was divided between 3 contexts, but was not weakened when the sessions took place in a single context. The results suggest that the use of multiple treatment settings in exposure-based therapies is only likely to reduce relapse if a sufficient number of sessions are provided in each of the treatment settings.     

Use of the ABA fear renewal paradigm to assess the effects of extinction with co-present fear inhibitors or excitors: Implications for theories of extinction and for treating human fears and phobias

In four experiments using albino rats in an ABA fear renewal paradigm, we studied conditioned fear in the A test context following extinction in Context B. Conditioned suppression of operant responding was the index of fear. In Experiments 1-3, we found that extinguishing a feared cue in compound with a putative conditioned inhibitor of fear led to more fear in the test context than did a conventional extinction procedure. In Experiments 4a and 4b, we found that extinguishing three feared cues in compound required one third the time and generally led to less fear to the cues in the test context than did the extinction of each cue separately. Thus, fear in the test context seems to vary inversely with the values of co-present cues during extinction in Context B. Results imply that cue value is actually reduced by extinction procedures rather than merely being opposed by a growing inhibitory process. Implications for theories of renewal and for clinical practice are discussed.     

Social transmission of Pavlovian fear: fear-conditioning by-proxy in related female rats

Pairing a previously neutral conditioned stimulus (CS; e.g., a tone) to an aversive unconditioned stimulus (US; e.g., a foot-shock) leads to associative learning such that the tone alone will elicit a conditioned response (e.g., freezing). Individuals can also acquire fear from a social context, such as through observing the fear expression of a conspecific. In the current study, we examined the influence of kinship/familiarity on social transmission of fear in female rats. Rats were housed in triads with either sisters or non-related females. One rat from each cage was fear conditioned to a tone CS+ shock US. On day two, the conditioned rat was returned to the chamber accompanied by one of her cage mates. Both rats were allowed to behave freely, while the tone was played in the absence of the foot-shock. The previously untrained rat is referred to as the fear-conditioned by-proxy (FCbP) animal, as she would freeze based on observations of her cage-mate’s response rather than due to direct personal experience with the foot-shock. The third rat served as a cage-mate control. The third day, long-term memory tests to the CS were performed. Consistent with our previous application of this paradigm in male rats (Bruchey et al. in Behav Brain Res 214(1):80–84, 2010), our results revealed that social interactions between the fear conditioned and FCbP rats on day two contribute to freezing displayed by the FCbP rats on day three. In this experiment, prosocial behavior occurring at the termination of the cue on day two was significantly greater between sisters than their non-sister counterparts, and this behavior resulted in increased freezing on day three. Our results suggest that familiarity and/or kinship influences the social transmission of fear in female rats.     

Heart rate reactivity in HAD and LAD rats during Pavlovian fear conditioning

Recently, we reported that High-Alcohol-Drinking (HAD) rats exhibited selective deficits in active avoidance learning under alcohol-naive conditions, and that administration of moderate doses of alcohol (0.5 and 1.0 g/kg) facilitated learning in these rats (Blankenship et al., 2000; Rorick et al., 2003b). We hypothesized that the deficits resulted from excessive fear in the aversive learning context and that the anxiolytic properties of alcohol may have contributed to the improved learning that was observed after alcohol administration. This hypothesis was supported by a recent study in which prolonged freezing in HAD rats was seen after a classical fear conditioning procedure (Rorick et al., 2003a). To provide additional evidence that HAD rats indeed exhibit behaviors consistent with the expression of increased fear in aversive learning contexts, we employed a Pavlovian fear conditioning task to measure heart rate in HAD and Low-Alcohol-Drinking (LAD) rats. In this study, HAD (HAD-1 and HAD-2) and LAD (LAD-1 and LAD-2) rats were assigned to one of three pre-exposure conditions: Context Only, Context/Tone, or Sequential (Context Only followed by Context/Tone) Pre-Exposure. Following pre-exposure, fear conditioning acquisition and extinction procedures were identical for all groups. Results indicated that although no baseline differences were observed between HAD and LAD rats, HAD rats receiving Context-Only pre-exposure exhibited excessive heart rate reactivity to the tone conditional stimulus during fear conditioning acquisition, compared to LAD rats receiving the same pre-exposure conditions. These findings support the hypothesis that HAD rats exhibit behaviors consistent with increased fear in aversive learning contexts, as measured by autonomic conditioning.     

Extinction and renewal of Pavlovian modulation in human sequential Feature Positive discrimination learning

Using a conditioned suppression task, we investigated extinction and renewal of Pavlovian modulation in human sequential Feature Positive (FP) discrimination learning. In Experiment 1, in context a participants were first trained on two FP discriminations, X-->A+/A- and Y-->B+/B-. Extinction treatment was administered in the acquisition context a (aaa group) or in a new context b (aba group), and comprised X-->A- extinction and Y- control trials. Discriminative X-->A/A responding was lost in both groups when tested in the extinction context, but partially recovered in the aba and not in the aaa group when tested in the acquisition context, suggesting extinction and renewal of extinguished modulation. The same was observed for the Y-->B/B control pair, however, questioning whether the loss of discriminative X-->A/A responding represented genuine extinction of modulation. In Experiment 2, including only aba groups, participants were trained in context a on two FP discriminations, X-->A+/A- and Y-->B+/B-, after which the group "Extinction" was exposed to X-->A- extinction trials in context b, whereas the group "Control" was exposed to X- control trials; concurrently, both groups received further Y-->B+/B- training. In the group Control, differential Y-->B/B and X-->A/A responding were acquired and maintained throughout the experiment. In the group Extinction, while Y-->B/B responding was also maintained throughout, differential X-->A/A responding disappeared because of X-->A- extinction treatment when tested in the extinction context b, but partially reappeared when tested in the acquisition context a. This evidences aba-renewal of extinguished modulation.     

US habituation,like CS extinction,produces a decrement in conditioned fear responding that is NMDA dependent and subject to renewal and reinstatement

Just as fear can be learned, it can also be inhibited. The most common way of reducing learned fear is through extinction, where the conditioned stimulus (CS) previously paired with an aversive unconditioned stimulus (US) is repeatedly presented on its own. Another, much less commonly studied, way to inhibit learned fear is by habituating, or devaluing, the US. In this procedure, fear responding to a CS is reduced by repeatedly presenting the US in the absence of the CS following the conditioning phase. The purpose of the present study was to directly compare the effects of US habituation and CS extinction on a learned fear response (freezing). Experiment 1 demonstrated that US habituation given either after (Experiment 1A) or before (Experiment 1B) fear conditioning reduced freezing to the CS at test. We then showed that the reduction in freezing resulting from either US habituation or CS extinction was context-specific (i.e., a change in context led to a renewal of the learned fear response; Experiment 2) and, furthermore, was attenuated when a pre-test shock was given (i.e., reinstatement of fear was observed in both cases; Experiment 3). Finally, Experiment 4 demonstrated that an injection of the NMDA antagonist MK-801 prior to US habituation impaired long-term retention of the learning that takes place during this procedure. Together, these results suggest that the decrement in conditioned fear responses produced by US habituation and CS extinction could rely on overlapping processes.     

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.     

A different recruitment of the lateral and basolateral amygdala promotes contextual or elemental conditioned association in Pavlovian fear conditioning

Convergent data suggest dissociated roles for the lateral (LA) and basolateral (BLA) amygdaloid nuclei in fear conditioning, depending on whether a discrete conditioned stimulus (CS)-unconditional stimulus (US) or context-US association is considered. Here, we show that pretraining inactivation of the BLA selectively impaired conditioning to context. In contrast, inactivation of the LA disrupted conditioning to the discrete tone CS, but also either impaired or enhanced contextual conditioning, depending on whether the context was in the foreground or in the background. Hence, these findings refine the current model of the amygdala function in emotional learning by showing that the BLA and the LA not only differentially contribute to elemental and context-US association, but also promote, through their interaction, the most relevant of these two associations.     

Exposure to a novel context after extinction causes a renewal of extinguished conditioned responses: Implications for the treatment of fear

Renewal gives an experimental model for the relapse of fear symptoms following exposure therapy. While renewal of extinguished fear in humans has been observed following a return to the original context in which fear was acquired (ABA design), it has been more difficult to show upon presentation of a novel context (ABC design). The present experiment used a particularly strong context manipulation in a fear conditioning procedure. Context was manipulated by using large photographs of real environments taken from various angles and was present throughout the entire experiment. A renewal of cognitive expectancy was found in both ABA and ABC renewal designs, although it was larger in the former than in the latter. Response times in making the expectancy judgments increased when there was a change to a new context. The results demonstrate consistency in fear renewal effects between human and animal studies and suggest that relapse following exposure therapy via renewal remains a danger when people encounter a previously feared object in a novel context.     

Reciprocal patterns of c-Fos expression in the medial prefrontal cortex and amygdala after extinction and renewal of conditioned fear

After extinction of conditioned fear, memory for the conditioning and extinction experiences becomes context dependent. Fear is suppressed in the extinction context, but renews in other contexts. This study characterizes the neural circuitry underlying the context-dependent retrieval of extinguished fear memories using c-Fos immunohistochemistry. After fear conditioning and extinction to an auditory conditioned stimulus (CS), rats were presented with the extinguished CS in either the extinction context or a second context, and then sacrificed. Presentation of the CS in the extinction context yielded low levels of conditioned freezing and induced c-Fos expression in the infralimbic division of the medial prefrontal cortex, the intercalated nuclei of the amygdala, and the dentate gyrus (DG). In contrast, presentation of the CS outside of the extinction context yielded high levels of conditioned freezing and induced c-Fos expression in the prelimbic division of the medial prefrontal cortex, the lateral and basolateral nuclei of the amygdala, and the medial division of the central nucleus of the amygdala. Hippocampal areas CA1 and CA3 exhibited c-Fos expression when the CS was presented in either context. These data suggest that the context specificity of extinction is mediated by prefrontal modulation of amygdala activity, and that the hippocampus has a fundamental role in contextual memory retrieval.Considerable interest has emerged in recent years in the neural mechanisms underlying the associative extinction of learned fear (Maren and Quirk 2004; Myers et al. 2006; Quirk and Mueller 2008). Notably, extinction is a useful model for important aspects of exposure-based therapies for the treatment of human anxiety disorders such as panic disorder and post-traumatic stress disorder (PTSD) (Bouton et al. 2001, 2006). During extinction, a conditioned stimulus (CS) is repeatedly presented in the absence of the unconditioned stimulus (US), a procedure that greatly reduces the magnitude and probability of the conditioned response (CR). After the extinction of fear, there is substantial evidence that extinction does not erase the original fear memory, but results in a transient inhibition of fear. For example, extinguished fear responses return after the mere passage of time (i.e., spontaneous recovery) or after a change in context (i.e., renewal) (Bouton et al. 2006; Ji and Maren 2007). In other words, extinguished fear is context specific. The return of fear after extinction is a considerable challenge for maintaining long-lasting fear suppression after exposure-based therapies (Rodriguez et al. 1999; Hermans et al. 2006; Effting and Kindt 2007; Quirk and Mueller 2008).In the last several years, considerable progress has been made in understanding the neural mechanisms underlying the context specificity of fear extinction. For example, lesions or inactivation of the hippocampus prevent the renewal of fear when an extinguished CS is presented outside of the extinction context (Corcoran and Maren 2001, 2004; Corcoran et al. 2005; Ji and Maren 2005, 2008; Hobin et al. 2006). In addition, neurons in the basolateral complex of the amygdala exhibit context-specific spike firing to extinguished CSs (Hobin et al. 2003; Herry et al. 2008), and this requires hippocampal input (Maren and Hobin 2007). Indeed, amygdala neurons that fire more to extinguished CSs outside of the extinction context are monosynaptically excited by hippocampal stimulation (Herry et al. 2008). In contrast, neurons that responded preferentially to extinguished CSs in the extinction context receive synaptic input from the medial prefrontal cortex (Herry et al. 2008).The prevalent theory of the interactions between the prefrontal cortex, hippocampus, and amygdala that lead to regulation of fear by context assumes that when animals experience an extinguished CS in the extinction context, the hippocampus drives prefrontal cortex inhibition of the amygdala to suppress fear (Hobin et al. 2003; Maren and Quirk 2004; Maren 2005). When animals encounter an extinguished CS outside of the extinction context, the hippocampus is posited to inhibit the prefrontal cortex and thereby promote amygdala activity required to renew fear. The hippocampus may also drive fear renewal through its direct projections to the basolateral amygdala (Herry et al. 2008). Although this model accounts for much of the extant literature on the context specificity of extinction, it is not known whether the nodes of this hypothesized neural network are coactive during the retrieval of fear and extinction memories. As a first step in addressing this issue, we used ex vivo c-Fos immunohistochemistry (e.g., Knapska et al. 2007) to generate a functional map of the neural circuits involved in the contextual retrieval of fear memory after extinction. Our results reveal reciprocal activity in prefrontal-amygdala circuits involved in extinction and renewal and implicate the hippocampus in hierarchical control of contextual memory retrieval within these circuits.     

Cholinergic modulation of Pavlovian fear conditioning in rats: differential effects of intrahippocampal infusion of mecamylamine and methyllycaconitine

The cholinergic system has consistently been implicated in Pavlovian fear conditioning. Considerable work has been done to localize specific nicotinic receptor subtypes in the hippocampus and determine their functional importance; however, the specific function of many of these subtypes has yet to be determined. An alpha7 nicotinic antagonist methyllycaconitine (MLA) (35 microg), and a broad spectrum non-alpha7 nicotinic antagonist mecamylamine (35 microg) was injected directly into the dorsal hippocampus or overlying cortex either 15 min pre-, 1 min post-, or 6h post-fear conditioning. One week after conditioning, retention of contextual and cue (tone) conditioning were assessed. A significant impairment in retention of contextual fear was observed when mecamylamine was injected 15 min pre- and 1 min post-conditioning. No significant impairment was observed when mecamylamine was injected 6h post-conditioning. Likewise, a significant impairment in retention of contextual fear was observed when MLA was injected 1 min post-conditioning; however, in contrast, MLA did not show any significant impairments when injected 15 min pre-conditioning, but did show a significant impairment when injected 6h post-conditioning. There were no significant impairments observed when either drug was injected into overlying cortex. No significant impairments were observed in cue conditioning for either drug. In general, specific temporal dynamics involved in nicotinic receptor function were found relative to time of receptor dysfunction. The results indicate that the greatest deficits in long-term retention (1 week) of contextual fear are produced by central infusion of MLA minutes to hours post-conditioning or mecamylamine within minutes of conditioning.     

Dental phobias and anxieties: evidence for conditioning processes in the acquisition and modulation of a learned fear

The present study investigated some of the factors which differentiate individuals with dental anxieties and phobias from those without such fears. In particular, two questions were addressed: (i) What differentiates subjects who have never been anxious about dental treatment from subjects who at some time have been anxious? and (ii) What factors lead to subjects changing their attitudes either from anxious to relaxed or from relaxed to anxious? The results suggest that the factors which influence the acquisition and modulation of dental anxieties are consistent with the associative and representational processes portrayed in contemporary models of human conditioning. Subjects who reported never having had anxieties about dental treatment were less likely to have had a painful dental treatment than subjects who did report an anxiety. Subjects who did report a painful dental experience but did not acquire anxiety reported a history of dental treatment favourable to the operation of latent inhibition. Subjects who reported that they were good at enduring pain were more likely to report a longer interval between their very first dental treatment and their first painful dental treatment. Under some conditions in which latent inhibition should have precluded the acquisition of a dental fear, an anxiety appeared to be acquired because a very painful experience had attenuated the latent inhibition process. Subjects whose dental anxiety did not remit reported significantly more painful and traumatic dental experiences than subjects whose anxiety did remit.     

Glutamate receptors in the medial geniculate nucleus are necessary for expression and extinction of conditioned fear in rats

Auditory fear conditioning requires anatomical projections from the medial geniculate nucleus (MGN) of the thalamus to the amygdala. Several lines of work indicate that the MGN is a critical sensory relay for auditory information during conditioning, but is not itself involved in the encoding of long-term fear memories. In the present experiments, we examined whether the MGN plays a similar role in the extinction of conditioned fear. Twenty-four hours after Pavlovian fear conditioning, rats received bilateral intra-thalamic infusions of either with NBQX (an AMPA receptor antagonist; Experiment 1) or MK-801 (an NMDA receptor antagonist; Experiment 1), anisomycin (a protein synthesis inhibitor; Experiment 2) or U0126 (a MEK inhibitor; Experiment 3) immediately prior to an extinction session in a novel context. The next day rats received a tone test in a drug-free state to assess their extinction memory; freezing served as an index of fear. Glutamate receptor antagonism prevented both the expression and extinction of conditioned fear. In contrast, neither anisomycin nor U0126 affected extinction. These results suggest that the MGN is a critical sensory relay for auditory information during extinction training, but is not itself a site of plasticity underlying the formation of the extinction memory.