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.     

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 voltage-gated calcium channel Cav1.3 mediates consolidation, but not extinction, of contextually conditioned fear in mice

Using pharmacological techniques, it has been demonstrated that both consolidation and extinction of Pavlovian fear conditioning are dependent to some extent upon L-type voltage-gated calcium channels (LVGCCs). Although these studies have successfully implicated LVGCCs in Pavlovian fear conditioning, they do not provide information about the specific LVGCC isoform involved. Both of the major LVGCC subtypes found in the brain (Cav1.2 and Cav1.3) are targets of the pharmacological manipulations used in earlier work. In this study, we used mice in which the gene for the pore-forming subunit (alpha1D) Cav1.3 was deleted (Cav1.3 knockout mice) to elucidate its contribution to consolidation and extinction of conditioned fear. We find that Cav1.3 knockout mice exhibit significant impairments in consolidation of contextual fear conditioning. However, once sufficiently overtrained, the Cav1.3 knockout mice exhibit rates of extinction that are identical to that observed in wild-type mice. We also find that Cav1.3 knockout mice perform as well as wild-type mice on the hidden platform version of the Morris water maze, suggesting that the consolidation deficit in conditioned fear observed in the Cav1.3 knockout mice is not likely the result of an inability to encode the context, but may reflect an inability to make the association between the context and the unconditioned stimulus.     

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.     

Divide and conquer: how object files adapt when a persisting object splits into two

Coherent visual experience requires not only segmenting incoming visual input into a structured scene of objects, but also binding discrete views of objects into dynamic representations that persist across time and motion. However, surprisingly little work has explored the principles that guide the construction and maintenance of such persisting object representations. What causes a part of the visual field to be treated as the same object over time? In the cognitive development literature, a key principle of object persistence is cohesion: An object must always maintain a single bounded contour. Here we demonstrate for the first time that mechanisms of adult midlevel vision are affected by cohesion violations. Using the object-file framework, we tested whether object-specific preview benefits-a hallmark of persisting object representations-are obtained for dynamic objects that split into two during their motion. We found that these preview benefits do not fully persist through such cohesion violations without incurring significant performance costs. These results illustrate how cohesion is employed as a constraint that guides the maintenance of object representations in adult midlevel vision.     

Effects of contingency violations on the extinction of a conditioned fear inhibitor and a conditioned fear excitor

Rats were used in a conditioned-suppression paradigm to assess the effects of contingency variations on responding to a conditioned inhibitor (CS-) and a conditioned excitor (CS+). In Experiment 1, various unconditioned stimulus (US) frequencies were equated across the presence and absence of a CS- in the context of either background cues (continuous-trial procedure) or an explicit neutral event (discrete-trial procedure). With both procedures, a CS-alone treatment enhanced inhibition, whereas treatments involving 50% or 100% reinforcement for the CS- eliminated inhibition without conditioning excitation to that CS. The latter outcome also occurred in Experiment 2, with discrete-trial training equating considerably reduced US frequencies for the presence and absence of the CS-. In further evidence that inhibition was eliminated without conditioning excitation to the CS-, Experiment 3 showed that a novel CS did not acquire excitation when 25%, 50%, or 100% reinforcement was equated across the presence and absence of that CS in the context of a discrete-trial event. Using the procedures of Experiment 1, Experiment 4 showed that a CS+ was extinguished by a CS-alone treatment but was substantially maintained by treatments involving 50% or 100% uncorrelated reinforcement. These effects for a CS+ and a CS- implicate CS-US contiguity, rather than contingency, as the factor determining the extinction of a CS.     

Interoceptive fear conditioning and panic disorder: the role of conditioned stimulus-unconditioned stimulus predictability

Interoceptive fear conditioning is at the core of contemporary behavioral accounts of panic disorder. Yet, to date only one study has attempted to evaluate interoceptive fear conditioning in humans (see Acheson, Forsyth, Prenoveau, & Bouton, 2007). That study used brief (physiologically inert) and longer-duration (panicogenic) inhalations of 20% CO(2)-enriched air as an interoceptive conditioned (CS) and unconditioned (US) stimulus and evaluated fear learning in three conditions: CS only, CS-US paired, and CS-US unpaired. Results showed fear conditioning in the paired condition, and fearful responding and resistance to extinction in an unpaired condition. The authors speculated that such effects may be due to difficulty discriminating between the CS and the US. The aims of the present study are to (a) replicate and expand this line of work using an improved methodology, and (b) clarify the role of CS-US discrimination difficulties in either potentiating or depotentiating fear learning. Healthy participants (N=104) were randomly assigned to one of four conditions: (a) CS only, (b) contingent CS-US pairings, (c) unpaired CS and US presentations, or (d) an unpaired "discrimination" contingency, which included an exteroceptive discrimination cue concurrently with CS onset. Electrodermal and self-report ratings served as indices of conditioned responding. Consistent with expectation, the paired contingency and unpaired contingencies yielded elevated fearful responding to the CS alone. Moreover, adding a discrimination cue to the unpaired contingency effectively attenuated fearful responding. Overall, findings are consistent with modern learning theory accounts of panic and highlight the role of interoceptive conditioning and unpredictability in the etiology of panic disorder.     

Reinstatement of fear to an extinguished conditioned stimulus: two roles for context

The authors studied the role of context in reinstatement. Freezing was reinstated when the conditioned stimulus (CS) was extinguished in 1 context and rats moved to another context for reexposure to the shock unconditioned stimulus (US) and test. It was also reinstated (rather than renewed) when rats were shocked in the extinction context and moved to another context for test. This reinstatement was CS specific and reduced by nonreinforced exposures to the extinction context. Rats shocked in the context in which a stimulus had been preexposed froze when tested in another context. These findings suggest 2 roles for context in reinstatement: conditioning of the test context (M. E. Bouton, 1993) and mediated conditioning by the extinction context (P. C. Holland, 1990).     

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.     

Sex,stress, and fear: Individual differences in conditioned learning

It has long been recognized that humans vary in their conditionability, yet the factors that contribute to individual variation in emotional learning remain to be delineated. The goal of the present study was to investigate the relationship among sex, stress hormones, and fear conditioning in humans. Forty-five healthy adults (22 females) underwent differential delay conditioning, using fear-relevant conditioned stimuli and a shock unconditioned stimulus. Salivary cortisol samples were taken at baseline and after acquisition training and a 24-h-delayed retention test. The results showed that acquisition of conditioning significantly correlated with postacquisition cortisol levels in males, but not in females. This sex-specific relationship was found despite similar overall levels of conditioning, unconditioned responding, and cortisol. There was no effect of postacquisition cortisol on consolidation of fear learning in either sex. These findings have implications for the understanding of individual differences in fear acquisition and risk factors for the development of affective disorders.     

Aversive conditioning in the rat: effects of a benzodiazepine and of an opioid agonist and antagonist on conditioned hypoalgesia and fear.

Hypoalgesia and fear co-occurred in rats trained on a heated floor and tested for their latencies to paw lick on that floor and to step down onto a nonheated floor. These responses were extinguished, suggesting a mediation by aversive conditioning processes. A benzodiazepine impaired the acquisition of aversive conditioning, but it did not attenuate the expression of conditioned hypoalgesia. The opioid agonist morphine also impaired acquisition across a range of drug doses and variations in hypoalgesic tolerance, whereas the opioid antagonist naloxone facilitated acquisition. The results are discussed in terms of the perceptual-defensive-recuperative (Fanselow, 1986) and working memory (Grau, 1987) models of the mechanisms for the co-occurrence of conditioned hypoalgesia and fear.     

Forgetting and conditioned suppression: role of a temporal discrimination

Several experiments have found complete retention of conditioned suppression when overall suppression to the signal for shock was measured. The present experiment examined retention of conditioned suppression but did so with a paradigm that produced temporal discrimination of shock occurrence. Nine rats were exposed to a flashing light signal of 5-min duration that always terminated with a shock. After several months, a temporal discrimination was well established, as shown by maximum suppression toward the end of the signal period. After remaining in the home cage for 25 days, the rats were again subjected to the conditioning procedure. The overall level of suppression remained the same but the temporal discrimination was not observed.     

Excitatory strength of expressive faces: effects of happy and fear expressions and context on the extinction of a conditioned fear response

In a recent study, Orr and Lanzetta (1984) showed that the excitatory properties of fear facial expressions previously described (Lanzetta & Orr, 1981; Orr & Lanzetta, 1980) do not depend on associative mechanisms; even in the absence of reinforcement, fear faces intensify the emotional reaction to a previously conditioned stimulus and disrupt extinction of an acquired fear response. In conjunction with the findings on acquisition, the failure to obtain extinction suggests that fear faces have some of the functional properties of "prepared" (fear-relevant) stimuli. In the present study we compared the magnitude of conditioned fear responses to happy and fear faces when a potent danger signal, the shock electrodes, are attached or unattached. If fear faces are functionally analogous to prepared stimuli, then, even in the absence of veridical support for an expectation of shock, they should retain excitatory strength, whereas happy faces should not. The results are consistent with this view of fear expressions. In the absence of reinforcement, and with shock electrodes removed, conditioned fear responses and basal levels of arousal were of greater magnitude for the fear-face condition than for the happy-face condition.     

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.     

Conditional forebrain deletion of the L-type calcium channel Ca V 1.2 disrupts remote spatial memories in mice

To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for remote memory consolidation, we generated conditional knockout mice in which the L-VGCC isoform Ca(V)1.2 was postnatally deleted in the hippocampus and cortex. In the Morris water maze, both Ca(V)1.2 conditional knockout mice (Ca(V)1.2(cKO)) and control littermates displayed a marked decrease in escape latencies and performed equally well on probe trials administered during training. In distinct contrast to their performance during training, Ca(V)1.2(cKO) mice exhibited significant impairments in spatial memory when examined 30 d after training, suggesting that Ca(V)1.2 plays a critical role in consolidation of remote spatial memories.     

Cardiac arrhythmias in the monkey during classically conditioned fear and excitement

Rhesus monkeys(Macaca mulatto) were tested in classic aversive and appetitive conditioning paradigms following complete coronary artery occlusion (CO) to test the hypothesis that “emotional stress” induces ventricular arrhythmias. Findings were based upon conditioning trials conducted for one or more weeks after occlusion in 13 animals. When all data from each animal for the week following CO were considered, there was no demonstrable tendency for arrhythmias to increase during “fear” conditioned to unavoidable electric shock or during “excitement” in anticipation of food. However, selected trials from six monkeys did reveal instances when changes in the frequency of occurrence of arrhythmias were coupled with behavioral conditioning. While analysis of these trials did not reveal any simple relationship between emotional stress and the development of ventricular arrhythmias after myocardial infarction, certain of the behavioral situations may be more potentially arrhythmogenic than others. For these selected trials, with respect to control, the number of arrhythmias may have increased or decreased upon presentation of the conditional stimulus; the exact response appears to depend upon the immediate physiologic status of the animal as well as on the behavioral condition. “More stressful” situations, such as aversive conditioning, are not necessarily associated with greater numbers of arrhythmias than were “less stressful” situations, such as appetitive conditioning. Arrhythmias appear to occur more frequently when an animal’s heart rate is within a given range; this may reflect underlying cardiac sympathetic and parasympathetic nerve activity.