Contextual fear-potentiated startle conditioning in humans: replication and extension

Contextual fear conditioning was examined using the startle reflex in two groups of participants over two sessions separated by 1/2 h. The conditioned stimulus (CS) was paired (paired group) or not (unpaired group) with an unpleasant shock during conditioning. The paired group showed conditioning to the CS that was well retained over the retention interval. Session I intertrial interval startles--a measure of contextual conditioning--were greater in the unpaired compared to the paired group. Context conditioning was retained in Session 2 and was present before the shock electrodes were attached. Self-rating of state anxiety, arousal, and pleasure indicated differential changes in mood from Session 1 to Session 2 in the two groups, with the unpaired group showing relatively greater negative affects compared to the paired group. These results indicate that unpredictable shocks lead to greater context conditioning as measured by startle and self-reports.     

Bilateral phosphorylation of ERK in the lateral and centrolateral amygdala during unilateral storage of fear memories

We previously showed that when rats were trained to fear an auditory conditioned stimulus (CS) by pairing it with a mild unilateral shock to the eyelid (the unconditioned stimulus, or US), conditioned freezing depended upon the amygdala contralateral but not ipsilateral from the US. It was proposed that convergent activation of amygdala neurons by the CS and US occurred mainly in the amygdala contralateral from US delivery, causing memories of the CS–US association to be stored primarily by that hemisphere. In the present study, we further tested this interpretation by administering unilateral infusions of U0126 (in 50% dimethyl sulfoxide (DMSO) vehicle) to block phosphorylation of extracellular signal-responsive kinase (ERK) in the amygdala prior to CS–US pairings. Conditioned freezing was impaired 24 h after training when U0126 was infused contralaterally—but not ipsilaterally—from the US, suggesting that fear memories were consolidated mainly by the contralateral amygdala. However, immunostaining experiments revealed that ERK phosphorylation was elevated in both hemispheres of the amygdale's lateral (LA) and centrolateral (CeL) nuclei after paired (but not unpaired (UNP)) presentations of the CS and US. Thus, fear acquisition induced ERK phosphorylation bilaterally in the amygdala, even though the ipsilateral hemisphere did not appear to participate in conditioned freezing. These findings suggest that associative plasticity may occur in both amygdala hemispheres even when only one hemisphere is involved in freezing behavior. Conditioning-induced ERK phosphorylation was identical in both hemispheres of LA, but was slightly greater in the contralateral than ipsilateral hemisphere of CeL. Hence, asymmetric induction of plasticity in CeL might help to explain why conditioned freezing depends preferentially upon the amygdala contralateral from the US in our fear conditioning paradigm.     

The basolateral amygdala determines the effects of fear memory on sleep in an animal model of PTSD

Fear conditioning [inescapable shock training (ST)] and fearful context re-exposure (CR) alone can produce significant fear indicated by increased freezing and reductions in subsequent rapid eye movement (REM) sleep. Damage to or inactivation of the basolateral nucleus of the amygdala (BLA) prior to or after ST or prior to CR generally has been found to attenuate freezing in the shock training context. However, no one has examined the impact of BLA inactivation on fear-induced changes in sleep. Here, we used the GABA_A agonist, muscimol (MUS), to inactivate BLA prior to ST, the period when fear is learned, and assessed sleep after ST and sleep and freezing after two CR sessions. Wistar rats (n = 14) were implanted with electrodes for recording sleep and with cannulae aimed bilaterally into BLA. After recovery, the animals were habituated to the injection procedure (handling) over 2 consecutive days and baseline sleep following handling was recorded. On experimental day 1, the rats were injected (0.5 μl) into BLA with either MUS (1.0 μM; n = 7) or vehicle (distilled water, n = 7) 30 min prior to ST (20 footshocks, 0.8 mA, 0.5-s duration, 60-s interstimulus interval). On experimental days 7 and 21, the animals experienced CR (CR1 and CR2, respectively) alone. Electroencephalogram and electromyogram were recorded for 8 h on each day, and the recording was scored for non-rapid eye movement sleep, REM sleep, and wakefulness. Freezing was examined during CR1 and CR2. MUS microinjections into BLA prior to ST blocked the post-training reduction in REM sleep seen in vehicle-treated rats. Furthermore, in MUS-treated rats, REM sleep after CR1 and CR2 was at baseline levels and freezing was significantly attenuated. Thus, BLA inactivation prior to ST blocks the effects of footshock stress on sleep and reduces fear memory, as indicated by the lack of freezing and changes in sleep after CR. These data indicate that BLA is an important regulator of stress-induced alterations in sleep and an important site for forming fear memories that can alter sleep.     

Methoxamine is an effective unconditioned stimulus for cardiovascular conditioning

New Zealand albino rabbits received classical conditioning training in which a 35-sec tone conditioned stimulus was paired with a bolus injection of methoxamine hydrochloride (Vasoxyl), an alpha 1-adrenergic agonist. Heart rate (HR) and blood pressure (BP) responses were recorded. Methoxamine produced a precipitous rise in BP and bradycardia as an unconditioned response (UR); pairings of tone and methoxamine over a 5-day period resulted in a gradually appearing tachycardia conditioned response (CR) which occurred shortly following tone onset. On the other hand, the BP CR was a pressor response. Accordingly, the HR CR was opposite in direction and, thus, apparently compensatory to the UR, whereas the BP CR was similar in direction to the UR. Neither of these cardiovascular changes were observed in control animals receiving either unpaired presentations of tone and methoxamine or tones paired with physiological saline. Most animals receiving either paired or unpaired infusions of methoxamine also showed consistent elevations in baseline HR as training progressed, relative to their respective day 1 levels, thus suggesting the development of compensatory HR CRs to the contextual cues associated with training.     

Fear conditioning and brain activity: a positron emission tomography study in humans

Regional cerebral blood flow (rCBF) was measured with H2 (15)O positron emission tomography in 8 healthy women before and after fear conditioning (i.e., paired shocks) and unpaired shocks to videotape cues. Conditioning was supported by enhanced peripheral nervous system recordings and subjective ratings. Fear conditioning increased rCBF in the central gray of the midbrain; bilaterally in the hypothalamus, the thalamus, and the left striatum; and in the right and left anterior cingulate and right prefrontal cortices. Regional CBF was attenuated bilaterally in the right and left prefrontal, temporal (including the amygdala), parietal, and occipital cortices, and in the left orbitofrontal cortex. When compared with unpaired shock presentations, fear conditioning resulted in elevated rCBF in the left cerebellum. Hence, in the present paradigm, only neural activity in the left cerebellum solely reflected processes associated with true Pavlovian conditioning.     

Olfactory-mediated fear conditioning in mice: simultaneous measurements of fear-potentiated startle and freezing

This study demonstrates that mice display olfactory-cued fear as measured with both freezing and fear-potentiated startle. Following a preconditioning test to measure any unconditioned responses to odor, mice received 5 pairings of a 10-s odor with a 0.25-s, 0.4-mA footshock. The next day, startle and freezing were measured in the presence and absence of the odor. Both fear measures increased after training with amyl acetate (Experiment 1) and acetophenone (Experiment 2). The enhancement of startle did not occur when the same number of odors and shocks were presented in an unpaired fashion (Experiment 3). Furthermore, mice were able to discriminate between an odor paired with shock and a nonreinforced odor (Experiment 4).     

Startle potentiation in rapidly alternating conditions of high and low predictability of threat

Effects of predictability of threat on potentiation of the startle reflex were investigated by presenting participants with predictable and unpredictable electric shocks. Shocks were presented either paired with a visual cue (paired condition) or unrelated to the presentation of the visual cues (unpaired condition). In contrast to previous slower-paced studies, conditions alternated at a rapid rate: each context lasted 8.5 s and within these contexts the visual cues had a duration of 1.5 s. Results replicated previous findings: in the predictable condition, startle responses were augmented by a threat-signaling stimulus, and startle responses in the unpredictable condition were larger than in a neutral condition in which no shocks were presented. In all three conditions, visual stimuli that did not carry information about when a shock could be presented augmented startle reactivity. A control experiment showed that the effects of threat on the startle response could not be ascribed to attention and that the effects of the lead stimuli that did not signal threat are likely to be unrelated to the effects of threat. These results show that the fear system is modulated dynamically as a function of rapidly changing information about threat and emphasize the role of predictability of an aversive stimulus in the distinction between cue-specific and contextual fear.     

Effects of cued and contextual fear on sleep in DBA/2J mice

Fear conditioning alters sleep, with the most consistent effect being significant reductions in rapid eye movement sleep (REM). DBA/2 (D2) mice show behavioral signs of anxiety, respond greater to shock training, and potentially behave differently in cued and contextual fear, raising the question of how fear conditioning would affect their sleep. D2 mice were implanted to record sleep via telemetry. After baseline sleep recording, groups of D2 mice were trained in cued (15 tone–shock pairings) and contextual (15 non-cued shocks) fear on 4 consecutive days. Cue and context control mice were given identical training but were never presented with shock. Sleep was recorded after shock training and after presentation of cue or context alone. Shock training produced selective suppression of REM. On the day after shock training was completed, light period sleep was significantly altered in mice in the cued fear group, but not in the contextual fear group. Subsequent presentation of fearful cues and re-exposure to the fearful contexts also produced significant reductions in REM. The effects of fear conditioning on sleep in D2 mice and other mouse strains and the differential effects of cued and contextual fear on sleep are discussed.     

Long-term effect of cued fear conditioning on REM sleep microarchitecture in rats

STUDY OBJECTIVES: To study long-term effects of conditioned fear on REM sleep (REMS) parameters in albino rats. DESIGN: We have investigated disturbances in sleep architecture, including muscle twitch density as REMS phasic activity, and freezing behavior in wakefulness, upon reexposure to a conditioned stimulus (CS) on Day 1 and Day 14 postconditioning. SUBJECTS: Male Sprague-Dawley rats prepared for polysomnographic recordings. INTERVENTIONS: After baseline sleep recording, the animals in the experimental group received five pairings of a 5-sec tone, co-terminating with a 1-sec, 1 mAfootshock. The control rats received similar numbers of tones and shocks, but explicitly unpaired. On postconditioning days, after reexposure to tones alone, sleep and freezing behavior were recorded. MEASUREMENTS AND RESULTS: Conditioned fear significantly altered REMS microarchitecture (characterized as sequential-REMS [seq-REMS: < or =3 min episode separation] and single-REMS [sin-REMS: >3 min episode separation]) on Day 14. The total amount and number of seq-REMS episodes decreased, while the total amount and number of sin-REMS episodes increased. Further, the CS induced significant increases in freezing and REMS myoclonic twitch density in the experimental group. Reexposure to the CS produced no alterations in controls. CONCLUSIONS: The results suggest that conditioned fear causes REMS alterations, including difficulty in initiating a REMS episode as indicated by the diminution in the number of seq-REMS episodes. Another finding, the increase in phasic activity, agrees with the inference from clinical investigations that retrieval of fearful memories can be associated with the long-term REMS disturbances characteristic of posttraumatic stress disorder.     

5-HT mechanisms of median raphe nucleus in the conditioned freezing caused by light/foot-shock association

We have shown that 5-HT mechanisms of the median raphe nucleus (MRN) are involved in contextual fear-conditioning processes as electrolytic or neurotoxic lesions with N-methyl-D-aspartate (NMDA) or injections of 8-hydroxy-2-(di-n-propilamino)-tetralin (8-OH-DPAT) into this structure inhibit freezing behavior in a contextual fear paradigm. In this work, we extend these studies by analyzing the behavioral responses in a classical fear-conditioning paradigm (light or tone/foot-shock association) in rats with either neurochemical lesion with NMDA or injected with 8-OH-DPAT into the MRN. The animals received NMDA or 8-OH-DPAT or saline microinjections into the MRN and were submitted to conditioning trials in an experimental chamber, where they received 10 foot-shocks (0.6 mA, 1 s, variable interval between 10 and 50 s) paired with tone or light (CS). On the next day, they were tested in a different experimental chamber, with or without CS presentation, where the duration of freezing and the number of rearing episodes were recorded. Light or tone alone caused a significant amount of freezing. NMDA lesions or 8-OH-DPAT injections into the MRN clearly inhibited freezing behavior in rats conditioned to light/foot-shock association, but not in the conditioning sessions with tones. Besides the proposed role in contextual fear conditioning, these results clearly show that MRN is involved in the fear conditioning with light as conditioned stimuli. Distinct neural substrates seem to subserve conditioning fear with acoustic stimuli.     

The effect of yohimbine on the extinction of conditioned fear: a role for context

Six experiments with rat subjects examined the effect of yohimbine, an alpha-2 adrenergic autoreceptor antagonist, on the extinction of conditioned fear to a tone. Experiments 1 and 2 demonstrated that systemic administration of yohimbine (1.0 mg/kg) facilitated a long-term decrease in freezing after extinction, and this depended on pairing drug administration with extinction training. However, Experiments 3 and 4 demonstrated that yohimbine did not eradicate the original fear learning: Freezing was renewed when the tone was tested outside of the extinction context. Experiments 5 and 6 found that the contextually specific attenuation of fear produced by yohimbine transferred to another extinguished conditional stimulus (CS) and not to a nonextinguished CS. The results suggest that yohimbine, when administered in the presence of a neutral context, creates a form of inhibition in that context that allows that specific context to reduce fear of an extinguished CS.     

The formation of auditory fear memory requires the synthesis of protein and mRNA in the auditory thalamus

The medial geniculate nucleus of the thalamus responds to auditory information and is a critical part of the neural circuitry underlying aversive conditioning with auditory signals for shock. Prior work has shown that lesions of this brain area selectively disrupt conditioning with auditory stimuli and that neurons in the medial geniculate demonstrate plastic changes during fear conditioning. However, recent evidence is less clear as to whether or not this area plays a role in the storage of auditory fear memories. In the current set of experiments rats were given infusions of protein or messenger RNA (mRNA) synthesis inhibitors into the medial geniculate nucleus of the thalamus 30 min prior to auditory fear conditioning. The next day animals were tested to the auditory cue and conditioning context. Results showed that rats infused with either inhibitor demonstrated less freezing to the auditory cue 24 h after training, while freezing to the context was normal. Autoradiography confirmed that the doses used were effective in disrupting synthesis. Taken together with prior work, these data suggest that the formation of fear memory requires the synthesis of new protein and mRNA at multiple brain sites across the neural circuit that supports fear conditioning.     

Fear-potentiated startle conditioning in humans: Explicit and contextual cue conditioning following paired versus unpaired training

Conditioned fear in response to explicit and contextual cues was examined using the startle reflex in three groups of participants over two sessions separated by 4–5 days. The conditioned stimulus (CS) was paired with an aversive unconditioned stimulus (US) (shock) during conditioning in the paired but not in the unpaired group. In the reaction time (RT) group, the US was a nonaversive visual signal for an RT task. In the paired group, the CS potentiated startle in the postconditioning phase. This conditioned response was fully retained over the retention interval. There was no substantial change in baseline startle (startle delivered in the absence of CS). By contrast, startle was not potentiated by the CS in the unpaired group, but baseline startle was increased from Session 1 to Session 2. In the RT group, startle was not affected by the CS, and baseline startle was reduced from Session 1 to Session 2. These results suggest that paired presentations of a CS and an aversive US result in conditioned fear in response to the CS but little contextual fear, whereas unpaired presentations of a CS and US leads to poor explicit cue conditioning but substantial contextual fear.     

Influence of massed and distributed context preexposure on contextual fear and Egr-1 expression in the basolateral amygdala

Preexposure to the conditioning context can influence the expression of context-conditioned fear. We used behavioral and early growth response gene (egr-1) assays in rats to study the effects of massed and distributed context preexposure on context-conditioned fear. The results demonstrated that massed context preexposure impaired acquisition of contextual fear, an effect here referred to as delayed shock deficit. Spaced context preexposure produced similar inhibitory effects. Significantly, the introduction of a brief change of context prior to conditioning completely reversed the deficit induced by massed, but not by distributed, context preexposure. This reversibility was inversely related to the duration of the context shift. The acquisition of context-conditioned fear was associated with enhanced Egr-1 expression in the basolateral amygdala (BLA). No such increase was evident in animals undergoing distributed context preexposure or in those experiencing massed preexposure without change of context. Remarkably, a brief change of context prior to conditioning not only facilitated learning following massed preexposure but also elicited a significant elevation of Egr-1 protein levels in the BLA. The findings shown demonstrated that the inhibitory effects of massed and distributed context preexposure on conditioning could be dissociable both behaviorally and physiologically. We suggest that the delayed shock deficit associated with massed preexposure derives from perceptual fade-out or inattention and its reversal by a brief change of context from attentional recovery.     

OPERANT CONDITIONING OF INCREASES IN BLOOD PRESSURE

Four monkeys were presented with 10 sec tones which terminated with shocks. The tones were immediately terminated without shock if the animal's diastolic blood pressure rose above a criterion level and remained high for 1 sec. Termination of the tone was followed by a 5 sec time out. Trials began whenever the pressure dropped below the criterion level. All subjects learned the avoidance task, showing diastolic elevations of up to 60 mm of mercury in response to the tones. A linear relationship was seen between the minimum pressure required for avoidance and the pressure achieved. No change in blood pressure accompanied a second stimulus which was never paired with shock. A fifth control monkey was yoked to one of the experimental monkeys and simultaneously received all shocks and tones as determined by the blood pressure of the experimental animal. The yoked control showed no pressure changes to the tones, but normal pressure elevations to shock.     

Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning.

The contribution of the amygdala and hippocampus to the acquisition of conditioned fear responses to a cue (a tone paired with footshock) and to context (background stimuli continuously present in the apparatus in which tone-shock pairings occurred) was examined in rats. In unoperated controls, responses to the cue conditioned faster and were more resistant to extinction than were responses to contextual stimuli. Lesions of the amygdala interfered with the conditioning of fear responses to both the cue and the context, whereas lesions of the hippocampus interfered with conditioning to the context but not to the cue. The amygdala is thus involved in the conditioning of fear responses to simple, modality-specific conditioned stimuli as well as to complex, polymodal stimuli, whereas the hippocampus is only involved in fear conditioning situations involving complex, polymodal events. These findings suggest an associative role for the amygdala and a sensory relay role for the hippocampus in fear conditioning.     

Effects of naltrexone and signaling inescapable electric shock on nociception and gastric lesions in rats

The antinociceptive effects of signaled shock and its physiological underpinnings were examined in two experiments. In Experiment 1, rats were exposed to one of three shock conditions: no shock, unsignaled shock, and signaled (by a 10-s, 1000-Hz tone) shock. In each condition the rats were tested hourly in the absence of tones for nociception, with vocalization to shock used as the behavioral measure. Rats receiving signaled shocks had stomach ulcer scores intermediate between those of no-shock and unsignaled shock animals. The signaled shock rats also displayed a pronounced vocalization antinociception effect. This suggested that signaled shock may be less aversive. Experiment 2 was designed to investigate a possible role of endogenous opiate peptides in these effects. In Experiment 2, animals received hourly injections of either the opiate antagonist naltrexone (7 mg/kg, ip) or saline. There were no significant effects of naltrexone on either stomach pathology or nociception scores. The same effects of signaled shock were obtained as in Experiment 1. It is concluded that the role of endogenous opiates in the effects of signaled shock seen here is minimal.     

Deterioration in learning and memory of fear conditioning in response to context in aged SAMP8 mice

This study examined age-dependent deficits in the learning and memory of fear conditioning, using a newly developed senescence-accelerated mouse (SAMP8) model of age-related brain dysfunction and its genetically related inbred strain (SAMR1). The mice were classically conditioned to tone by giving aversive foot shocks in a distinct experimental box (context). After conditioning, fear in response to the original context without the tone and to the tone in a different context were tested with no shocks. Freezing behavior was used as a reliable index of fear. At 4 and 8 months, contextual fear was weaker in the accelerated senescence-prone SAMP8 mice than in the accelerated senescence-resistant SAMR1 mice. However, at 1 and 2 months, both SAMP8 and SAMR1 mice showed significant contextual fear to equivalent levels. Aging did not affect the fear response to tone. These results indicate that SAMP8 mice have age-related learning and memory deficits in their fear response evoked by contextual but not explicit tone stimuli. Age-related hippocampal dysfunction is suggested to be the cause of these age-related deficits in contextual fear conditioning in SAMP8 mice.     

Hippocampal and amygdalar involvement in discriminatory place learning

A conflict task was developed that simultaneously examines place aversion learning and fear-motivated context discrimination. The task superimposed Pavlovian discriminative fear conditioning on an appetitively motivated instrumental response (alternation). Rats were trained to alternate along a high-walled, diamond-shaped runway between two chambers for food. On half of the trials, a tone CS signaled the fact that a fixed section at the apex of the runway was electrified. Both the tone and the shock were turned on at the beginning of, and remained on for the duration of, each tone trial. A new trial was initiated at the time the animal entered the subsequent food chamber. Therefore, during a tone trial, in order to attain additional food reinforcement, the animal had to cross over the electrified region at the runway apex. Behavioral performance of rats with small lesions of the amygdala or dorsal hippocampus (DH) was compared with that of sham-operated controls. All groups displayed significant discriminative responding, hesitating more on tone trials while in areas of the runway adjacent to the shock region. Animals with lesions of the DH were similar to controls with respect to the tone-mediated discrimination, yet were delayed in the initial expression of a location-specific fear response. Conversely, amygdala lesions did not affect place learning; however, these animals were impaired in their suppression of the fear response following repeated unpaired trials.     

Preexposure to (un)predictable shock modulates discriminative fear learning between cue and context: an investigation of the interaction between fear and anxiety

It has been suggested that prior experiences with unpredictable/uncontrollable stressors facilitate subsequent fear learning and the development of anxiety disorders. However, animal research documents that preexposure to unpredictable stressors (USs) impede later fear conditioning with that US. These differential predictions were tested in a human experimental model of clinical anxiety. One (US-only) group was preexposed to unpredictable shocks, a second (Unpaired) group received explicitly unpaired presentations of a neutral shape and the shock, and a third (Paired) group received paired shape-shock presentations. Next, all groups received training with a novel shape, using the same shock (50% reinforcement). Fear responding was assessed through startle modulation and online shock-expectancy ratings. Results showed retarded fear learning in the unpredictable groups compared to the predictable group. We argue that prior experiences of unpredictability may still contribute to the development of clinical anxiety, by impeding adaptive fear learning and perpetuating the perception of unpredictability/uncontrollability.