Associative fear conditioning of enkephalin mRNA levels in central amygdalar neurons

The central nucleus of the amygdala (CEA) is required for the expression of learned fear responses. This study used in situ hybridization to show that mRNA levels of the neuropeptide enkephalin are increased in CEA neurons after rats are placed in an environment that they associate with an unpleasant experience. In contrast, mRNA levels of another neuropeptide, corticotropin releasing hormone, do not change under the same conditions in the CEA of the same rats. Conditioned neuropeptide levels in amygdalar circuits may act as a reversible "gain control" for long-term modulation of subsequent fear responses.     

Unconscious amygdalar fear conditioning in a subset of chronic fatigue syndrome patients

Here, a novel hypothesis for chronic fatigue syndrome (CFS) is proposed. CFS may be a neurophysiological disorder focussing on the amygdala. During a 'traumatic' neurological event often involving acute psychological stress combined with a viral infection or other chemical or physiological stressor, a conditioned network or 'cell assembly' may be created in the amygdala. The unconscious amygdala may become conditioned to be chronically sensitised to negative symptoms arising from the body. Negative signals from the viscera or physiological, chemical and dietary stressors, become conditioned stimuli and the conditioned response is a chronic sympathetic outpouring from the amygdala via various brain pathways including the hypothalamus. This cell assembly then produces the CFS vicious circle, where an unconscious negative reaction to symptoms causes immune reactivation/dysfunction, chronic sympathetic stimulation, leading to sympathetic dysfunction, mental and physical exhaustion, and a host of other distressing symptoms and secondary complications. And these are exactly the symptoms that the amygdala and associated limbic structures are trained to monitor and respond to, perpetuating a vicious circle. Recovery from CFS may involve projections from the medial prefrontal cortex to the amygdala, to control the amygdala's expressions. I shall firstly discuss predisposing, precipitating, and perpetuating factors involved in the possible etiology of chronic fatigue syndrome (CFS), followed by the patient's experience of the illness. Finally, I shall look at a suggested explanation for the symptoms of CFS.     

Impaired fear memories are correlated with subregion-specific deficits in hippocampal and amygdalar LTP

Inbred mouse strains have different genetic backgrounds that likely influence memory and long-term potentiation (LTP). LTP, a form of synaptic plasticity, is a candidate cellular mechanism for some forms of learning and memory. Strains with impaired fear memory may have selective LTP deficits in different hippocampal subregions or in the amygdala. The authors assessed fear memory in 4 inbred strains: C57BL/6NCrlBR (B6), 129S1/SvImJ (129), C3H/HeJ (C3H), and DBA/2J (D2). The authors also measured LTP in the hippocampal Schaeffer collateral (SC) and medial perforant pathways (MPP) and in the basolateral amygdala. Contextual and cued fear memory, and SC and amygdalar LTP, were intact in B6 and 129, but all were impaired in C3H and D2. MPP LTP was similar in all 4 strains. Thus, SC, but not MPP, LTP correlates with hippocampus-dependent contextual memory expression, and amygdalar LTP correlates with amygdala-dependent cued memory expression, in these inbred strains.     

Effect of hippocampal lesions on heart-rate during classical fear conditioning

Of 22 male albino rats, eight received bilateral lesions at three levels of the hippocampus, six received bilateral lesions of the amygdaloid complex and eight served as unoperated controls. Each rat received 30 fear conditioning trials in which shock was paired with a CS, either light or a tone. After every tenth trial there was a test trial in which heart-beats were recorded. The typical heart-rate response of controls was deceleration-acceleration while for hippocampal lesioned rats it was short latency acceleration. The results were interpreted in terms of two heart-rate theories.     

Effects of psilocybin on hippocampal neurogenesis and extinction of trace fear conditioning

Drugs that modulate serotonin (5-HT) synaptic concentrations impact neurogenesis and hippocampal (HPC)-dependent learning. The primary objective is to determine the extent to which psilocybin (PSOP) modulates neurogenesis and thereby affects acquisition and extinction of HPC-dependent trace fear conditioning. PSOP, the 5-HT_2A agonist 25I-NBMeO and the 5-HT_2A/C antagonist ketanserin were administered via an acute intraperitoneal injection to mice. Trace fear conditioning was measured as the amount of time spent immobile in the presence of the conditioned stimulus (CS, auditory tone), trace (silent interval) and post-trace interval over 10 trials. Extinction was determined by the number of trials required to resume mobility during CS, trace and post-trace when the shock was not delivered. Neurogenesis was determined by unbiased counts of cells in the dentate gyrus of the HPC birth-dated with BrdU co-expressing a neuronal marker. Mice treated with a range of doses of PSOP acquired a robust conditioned fear response. Mice injected with low doses of PSOP extinguished cued fear conditioning significantly more rapidly than high-dose PSOP or saline-treated mice. Injection of PSOP, 25I-NBMeO or ketanserin resulted in significant dose-dependent decreases in number of newborn neurons in hippocampus. At the low doses of PSOP that enhanced extinction, neurogenesis was not decreased, but rather tended toward an increase. Extinction of “fear conditioning” may be mediated by actions of the drugs at sites other than hippocampus such as the amygdala, which is known to mediate the perception of fear. Another caveat is that PSOP is not purely selective for 5-HT_2A receptors. PSOP facilitates extinction of the classically conditioned fear response, and this, and similar agents, should be explored as potential treatments for post-traumatic stress disorder and related conditions.     

Sleep deprivation increases threat beliefs in human fear conditioning

Sleep disturbances and anxiety disorders exhibit high comorbidity levels, but it remains unclear whether sleep problems are causes or consequences of increased anxiety. To experimentally probe the aetiological role of sleep disturbances in anxiety, we investigated in healthy participants how total sleep deprivation influences fear expression in a conditioning paradigm. In a fear conditioning procedure, one face stimulus (conditioned stimulus [CS+]) was paired with electric shock, whereas another face stimulus was not (unpaired stimulus [CS?]). Fear expression was tested the next morning using the two face stimuli from the training phase and a generalization stimulus (i.e. a morph between the CS+ and CS? stimuli). Between fear conditioning and test, participants were either kept awake in the laboratory for 12 hr (n = 20) or had a night of sleep at home (n = 20). Irrespective of stimulus type, subjective threat expectancies, but not skin conductance responses, were enhanced after sleep deprivation, relative to regular sleep. These results suggest that sleep disturbances may play a role in anxiety disorders by increasing perceived threat.     

Nicotine enhances contextual fear conditioning and ameliorates ethanol-induced deficits in contextual fear conditioning

Nicotine and ethanol are 2 commonly used and abused drugs that have divergent effects on learning. The present study examined the effects of acute nicotine (0.25 mg/kg), ethanol (1.0 g/kg), and ethanol-nicotine coadministration on fear conditioning in C57BL/6 mice. Mice were assessed for contextual and cued fear conditioning at 1 day and 1 week posttraining. Ethanol disrupted acquisition but not consolidation of contextual fear conditioning; nicotine enhanced contextual fear conditioning and ameliorated ethanol-associated deficits in contextual fear conditioning. Mecamylamine antagonized this effect. Fear conditioning was reassessed 1 week after initial testing with no drug administered. At the 1-week retest, mice previously treated with nicotine continued to show enhanced contextual fear, and mice previously treated with ethanol continued to show contextual fear deficits. Thus, nicotine both produces a long-lasting enhancement of contextual fear conditioning and protects against ethanol-associated deficits.     

Contextual fear conditioning is associated with lateralized expression of the immediate early gene c-fos in the central and basolateral amygdalar nuclei

Fos, the protein product of the immediate early gene c-fos, was used to map functional circuitry underlying contextual conditioned fear. Male rats were given footshocks in a distinctive context and later tested using freezing as the behavioral measure and compared with no-shock and no-retention-test control groups. An increased number of Fos-immunoreactive neurons was found in the lateral part of the central nucleus and in the anterior basolateral and lateral amygdalar nuclei in the brains of the conditioned-fear group compared with controls. Further, a greater number of Fos-immunoreactive neurons was observed in the right central and anterior basolateral nuclei compared with the number of labeled neurons in these structures on the left.     

Repeated restraint stress facilitates fear conditioning independently of causing hippocampal CA3 dendritic atrophy

This study investigated whether 21 days of restraint stress (6 hr/day) and the subsequent hippocampal dendritic atrophy would affect fear conditioning, a memory task with hippocampal-dependent and hippocampal-independent components. Restraint-stressed rats were injected daily (21 days) with tianeptine (10 mg/kg; to prevent hippocampal atrophy) or vehicle then tested on fear conditioning (Days 23-25, with 2 tone-shock pairings) and open field (Day 25). Restraint stress enhanced freezing to context (hippocampal-dependent behavior) and tone (hippocampal-independent) and decreased open-field exploration, irrespective of whether tianeptine was given. Results confirmed that stress produced CA3 dendritic atrophy and tianeptine prevented it. Moreover, CA3 dendritic atrophy was not permanent but reversed to control levels by 10 days after the cessation of restraint stress. These data argue that different neural substrates underlie spatial recognition memory and fear conditioning.     

Amygdala-dependent and amygdala-independent pathways for contextual fear conditioning

The basolateral amygdala (BLA), consisting of the lateral and basal nuclei, is considered to be essential for fear learning. Using a temporary inactivation technique, we found that rats could acquire a context-specific long-term fear memory without the BLA but only if intensive overtraining was used. BLA-inactivated rats' learning curves were characterized by slow learning that eventually achieved the same asymptotic performance as rats with the BLA functional. BLA inactivation abolished expression of overtrained fear when rats were overtrained with a functional BLA. However, BLA-inactivation had no effect on the expression of fear in rats that learned while the BLA was inactivated. These data suggest that there are primary and alternate pathways capable of mediating fear. Normally, learning is dominated by the more efficient primary pathway, which prevents learning in the alternate pathway. However, alternate pathways compensate when the dominant pathway is compromised.     

Sex differences in contextual fear conditioning are associated with differential ventral hippocampal extracellular signal-regulated kinase activation

Although sex differences have been reported in hippocampal-dependent learning and memory, including contextual fear memories, the underlying molecular mechanisms contributing to such differences are not well understood. The present study examined the extent to which sex differences in contextual fear conditioning are related to differential activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), a protein kinase critically involved in memory formation. We first show that male rats exhibit more long-term retention of contextual fear conditioning than female rats. During a tone test, females spent more time freezing than males, although both sexes exhibited robust retention of auditory fear learning. Using Western blot analysis, we then show that phosphorylated ERK levels in ventral, but not dorsal, hippocampus are higher in males than females, relative to same-sex controls, 60 minutes after fear conditioning. Post-conditioning increases in ERK activation were observed in the amygdala in both males and females, suggesting a selective effect of sex on hippocampal ERK activation. Together, these findings suggest that differential activation of the ERK signal transduction pathway in male and female rats, particularly in the ventral hippocampus, is associated with sex differences in contextual fear.     

Antisense DNA against calcineurin facilitates memory in contextual fear conditioning by lowering the threshold for hippocampal long-term potentiation induction

In the previous study, we demonstrated that the antisense oligodeoxynucleotides against calcineurin Aalpha and Abeta, catalytic subunits of Ca(2+)/calmodulin-dependent protein phosphatase, produce a facilitatory effect on long-term potentiation induction in the hippocampal CA1 region in rats anesthetized with urethane. Here, we have studied how animals, in which the hippocampal long-term potentiation induction is enhanced by antisense oligodeoxynucleotides against calcineurin, perform in learning tasks that depend on hippocampal function. The rats received antisense oligodeoxynucleotides by bilateral ventricular administration via miniosmotic pumps. We tested four groups of rats, three infused with either antisense oligodeoxynucleotides, scramble oligodeoxynucleotides, or saline, and untreated rats, for two types of hippocampus-dependent learning, water maze and contextual fear conditioning. After the behavioral tests, we conducted a long-term potentiation induction test to determine whether long-term potentiation induction was enhanced. In contextual fear conditioning, rats in which long-term potentiation induction was enhanced by antisense oligodeoxynucleotides displayed significantly more conditioned freezing response than control rats. Rats with enhanced long-term potentiation induction showed no differences in shock sensitivity, general activity, or light-dark choice from control rats. In contrast with contextual fear conditioning, rats with enhanced long-term potentiation induction showed no difference in spatial learning performance on the water maze compared with control rats.These results demonstrate that an enhancement in long-term potentiation induction produced by the inhibition of calcineurin leads to an increase in memory strength in specific forms of hippocampus-dependent learning.     

Appetitive-aversive interactions in Pavlovian fear conditioning

The existence of value coding and salience coding neurons in the mammalian brain, including in habenula and ventral tegmental area, has sparked considerable interest in the interactions that occur between Pavlovian appetitive and aversive conditioning. Here we studied these appetitive-aversive interactions at the behavioral level by assessing the learning that occurs when a Pavlovian appetitive conditioned stimulus (conditional stimulus, CS) serves as a CS for shock in Pavlovian fear conditioning. A Pavlovian appetitive CS was retarded in the rate at which it could be transformed into a fear CS (counterconditioning), but the presence of the appetitive CS augmented fear learning to a concurrently presented neutral CS (superconditioning). Retardation of fear learning was not alleviated by manipulations designed to restore the associability of the appetitive CS before fear conditioning but was alleviated by manipulations designed to increase the aversive quality of the shock unconditioned stimulus (US). These findings are consistent with opponent interactions between the appetitive and aversive motivational systems and provide a behavioral approach for assessing the neural correlates of these appetitive-aversive interactions.     

Mesolimbic dopaminergic pathways in fear conditioning

One of the most common paradigms used to study the biological basis of emotion, as well as of learning and memory, is Pavlovian fear conditioning. In the acquisition phase of a fear conditioning experiment, an emotionally neutral conditioned stimulus (CS)--which can either be a discrete stimulus, such as a tone, or a contextual stimulus, such as a specific environment--is paired with an aversive unconditioned stimulus (US), for example a foot shock. As a result, the CS elicits conditioned fear responses when subsequently presented alone during the expression phase of the experiment. While considerable work has been done in relating specific circuits of the brain to fear conditioning, less is known about its regulation by neuromodulators; the understanding of which would be of therapeutic relevance for fear related diseases such as phobia, panic attacks, post traumatic stress disorder, obsessive compulsive disorder, or generalized anxiety disorder. Dopamine is one of the neuromodulators most potently acting on the mechanisms underlying states of fear and anxiety. Recently, a growing body of evidence has suggested that dopaminergic mechanisms are significant for different aspects of affective memory, namely its formation, expression, retrieval, and extinction. The aim of this review is to clarify the complex actions of dopamine in fear conditioning with respect to the wide-spread distribution of dopaminergic innervation over structures constituting the fear related circuitry. A particular effort is made to understand how dopamine in the amygdala, medial prefrontal cortex and nucleus accumbens--target structures of the mesolimbic dopamine system originating from the ventral tegmental area--could relate to different aspects of fear conditioning.     

Nicotine and extinction of fear conditioning

Despite known health risks, nicotine use remains high, especially in populations diagnosed with mental illnesses, including anxiety disorders and Post-Traumatic Stress Disorder (PTSD). Smoking in these populations may relate to the effects of nicotine on emotional memories. The current study examined the effects of nicotine administration on the extinction of conditioned fear memories. C57BL/6J mice were trained with two white noise conditioned stimulus (CS; 30 s, 85 dB)–foot shock (2 s, 0.57 mA) pairings. Extinction sessions consisted of six presentations of the CS (60 s) across multiple days. Mice were either tested in an AAA design, in which all stages occurred in the same context, or in an ABA design to identify if context changes alter extinction. Saline or nicotine was administered 5 min before training and/or extinction. In the AAA design, nicotine administration before training did not alter extinction. Nicotine administered prior to extinction sessions enhanced extinction and nicotine administered before training and extinction decreased extinction. In the ABA design, nicotine administered before extinction enhanced extinction and blocked context renewal of conditioned fear, while nicotine administered during training and extinction did not alter extinction but enhanced the context renewal of conditioned fear. Nicotine has a differential effect on extinction of fear conditioning depending on when it is administered. Administration during extinction enhances extinction whereas administration during training and extinction may strengthen contextual fear memories and interfere with extinction.     

Learning to fear suffocation: a new paradigm for interoceptive fear conditioning

The present study aimed to establish a new interoceptive fear conditioning paradigm. The conditioned stimulus (CS) was a flow resistor that slightly obstructs breathing; the unconditional stimulus (US) was a breathing occlusion. The paired group (N = 21) received 6 acquisition trials with paired CS–US presentations. The unpaired group (N = 19) received 6 trials of unpaired CS–US presentations. In the extinction phase, both groups were administered 6 CS‐only trials. Measurements included startle eyeblink response, electrodermal responses, and self‐reported US expectancy. In the paired group, startle blink responses were larger during CS compared to intertrial interval during acquisition and extinction. Electrodermal and US expectancies were larger for the paired than for the unpaired group during acquisition, but not during extinction. The present paradigm successfully established interoceptive fear conditioning with panic‐relevant stimuli.     

Effects of prolonged exposure to context following contextual fear conditioning on synaptic properties in rat hippocampal slices

Acute stressful events enhance plasma corticosterone release and profoundly affect synaptic functions, which are involved in the development of stress-related cognitive and mental disorders. However, how exposure to stressful context immediately after stress further modulates the physiological responses is not fully understood. Here, we found that acute stress inhibited paired-pulse facilitation in hippocampal slices of Wistar rats which were subjected to contextual fear conditioning. But such inhibition was reversed by subsequent prolonged exposure to foot-shock context or returning to home cage for 1 h. Interestingly, foot-shock stress-facilitated LTD induced by low frequency stimulation (LFS, 900 pulses at 1 Hz) was maintained by subsequent exposure to foot-shock context but was reversed by returning to home cage environment. Moreover, plasma corticosterone level was still kept higher in rats exposed to foot-shock context but not to home cage. Findings suggest that remaining in stressful environment immediately after stress maintains acute stress-facilitated LTD and higher level of neuroendocrine response. Our results also contribute to further understanding the critical role of timely intervention in mediating stress-related aversive changes in human.     

P50 suppression in human discrimination fear conditioning paradigm using danger and safety signals

Auditory P50 suppression, which is assessed using a paired auditory stimuli (S1 and S2) paradigm to record the P50 mid-latency evoked potential, is assumed to reflect sensory gating. Recently, P50 suppression deficits were observed in patients with anxiety disorders, including panic disorder, post-traumatic stress disorder and obsessive-compulsive disorder, as we previously reported. The processes of fear conditioning are thought to play a role in the pathophysiology of anxiety disorders. In addition, we found that the P50 sensory gating mechanism might be physiologically associated with fear conditioning and extinction in a simple human fear-conditioning paradigm that involved a light signal as a conditioned stimulus (CS+). Our objective was to investigate the different patterns of P50 suppression in a discrimination fear-conditioning paradigm with both a CS+ (danger signal) and a CS- (safety signal). Twenty healthy volunteers were recruited. We measured the auditory P50 suppression in the control (baseline) phase, in the fear-acquisition phase, and in the fear-extinction phase using a discrimination fear-conditioning paradigm. Two-way (CSs vs. phase) Analysis of variance with repeated measures demonstrated a significant interaction between the two factors. Post-hoc LSD analysis indicated that the P50 S2/S1 ratio in the CS+ acquisition phase was significantly higher than that in the CS- acquisition phase. These results suggest that the auditory P50 sensory gating might differ according to the cognition of the properties (potentially dangerous or safe) of the perceived signal.     

Contextual fear extinction ameliorates sleep disturbances found following fear conditioning in rats

STUDY OBJECTIVE: To examine the effects of fear extinction on subsequent sleep in rats and to compare it with the effects seen following contextual reminders of fear. DESIGN: Habituation of the rats to handling and baseline recordings were obtained over 2 consecutive days. Afterward, the rats were subjected to shock training (ST; day 1), context reexposure (CR; either 30 or 60 min; day 2), and fear recall (R; day 3). Percentage time spent in freezing (FT%) was observed during ST, CR, and R exposures. Sleep was recorded for 20 h (8-h light and 12-h dark period) following ST, CR, and R. SETTING: NA SUBJECTS: The subjects were outbred Wistar rats randomly assigned to one of two groups: contextual fear (FR; n = 7) or contextual extinction (EXT; n = 7). INTERVENTIONS: The rats were surgically implanted with electrodes for recording the electroencephalogram and electromyogram for determining arousal state. MEASUREMENTS AND RESULTS: There were no differences between groups on FT% during ST or the first 30 min of CR; however, during R, the FR group had greater FT% than EXT. Sleep did not differ between groups following ST. Following CR, EXT exhibited significantly more total sleep, NREM, and REM than FR. After R, there were no differences between groups. CONCLUSIONS: Rats that exhibit extinction of contextual fear show significantly increased sleep compared to rats who continue to exhibit contextual fear. This suggests that sleep disturbances normally experienced in humans following traumatic events or reminders may be ameliorated by therapies that address and eliminate the associated fear.