The central amygdala is critical for the acquisition and expression of fear memories. This region receives a dense innervation from brainstem noradrenergic cell groups and has a high level of α2-adrenoceptor expression. Using whole-cell electrophysiological recordings from rat brain slices, we characterise the role of pre-synaptic α2-adrenoceptor in modulating discrete inhibitory and excitatory connections within both the lateral and medial division of the central amygdala. The selective α2-adrenoceptor agonist clonidine blocked the excitatory input from the pontine parabrachial neurons onto neurons of the lateral central amygdala. In addition, clonidine blocked inhibitory connections from the medial paracapsular intercalated cell mass onto both lateral and medial central amygdala neurons. To examine the behavioural consequence of α2-adrenoceptor-mediated inhibition of these inputs, we infused clonidine into the central amygdala prior to contextual fear-conditioning. In contrast to vehicle-infused rats, clonidine-infused animals displayed reduced levels of freezing 24 hours after training, despite showing no difference in freezing during the training session. These results reveal a role for α2-adrenoceptors within the central amygdala in the modulation of synaptic transmission and the formation of fear-memories. In addition, they provide further evidence for a role of the central amygdala in fear-memory formation.

Evaluative conditioning (EC) refers to a change in liking of a conditioned stimulus (CS) subsequent to its repeated pairing with a valent stimulus (US). Two studies that bring new light on the highly debated question of the role of awareness in EC were conducted. We developed an innovative method motivated by higher order and integration theories of consciousness to distinguish between the role of conscious and unconscious knowledge about the pairings. On each trial of the awareness test, participants had to indicate the valence of the US associated with a given CS and to make a 'structural knowledge attribution' by reporting the basis of their response. Valence identification accuracy was used to evaluate knowledge while the knowledge attribution was used to measure the conscious status of knowledge. Memory attribution indicated conscious knowledge about the pairings while feeling-based and random attributions indicated unconscious knowledge. A meta-analysis of the two studies revealed that valence identification accuracy was above chance level for memory and feeling-based attributions but not for the random attribution. EC was found in the three attributions. While EC effect size was medium for the memory attribution it was small for feeling-based and random attributions. Moreover, Experiment 2 included a delayed test. EC was still present 24 h after the conditioning took place. The results obtained for memory and feeling-based attributions suggest that both conscious and unconscious knowledge may underlie EC. The results obtained for random attribution suggest that EC may also occur without any knowledge of US valence.

The timing of voluntary exercise relative to drug conditioning is important to its "neuroprotective" effects, though it is unclear whether the voluntary exercise needs to occur temporally contiguous with drug conditioning, or occur during an early, developmental period, but non-contiguous with drug conditioning, for its "neuroprotective" effects. To distinguish between these two ideas, the timing of voluntary exercise relative to drug conditioning on the development and extinction of conditioned hyperactivity, and induction of sensitization was manipulated in the present experiment. Specifically, half of the exercise mice were permitted access to home-cage running wheels for 6 continuous weeks (Exercise-Exercise) whereas the other half of the exercise mice were permitted access to home-cage running wheels only for the first 3  weeks and then had the wheels removed (Exercise-Sedentary). Likewise, half of the sedentary mice had no home-cage running wheels for 6 continuous weeks (Sedentary-Sedentary) whereas the other half of the sedentary mice were permitted access to the home-cage running wheels for the last 3 weeks prior to the acquisition phase (Sedentary-Exercise). Mice received vehicle or methamphetamine (Meth; 1.0 mg/kg; acquisition), followed by saline-alone sessions (extinction) and finally challenged with an escalating Meth-regimen (0.25 → 1.0 mg/kg). While all Meth-paired groups, regardless of exercise regimen, showed conditioned hyperactivity, Exercise-Exercise and Exercise-Sedentary mice were less responsive to chronic Meth exposure and showed slower extinction compared to the other Meth-paired groups. These results suggest an early exercise regimen, during a critical developmental window, protects against the stimulant properties of Meth and simultaneously facilitates contextual learning.

The honey bee has been an important model organism for studying learning and memory. More recently, the honey bee has become a valuable model to understand perception and cognition. However, the techniques used to explore psychological phenomena in honey bees have been limited to only a few primary methodologies such as the proboscis extension reflex, sting extension reflex, and free flying target discrimination-tasks. Methods to explore operant conditioning in bees and other invertebrates are not as varied as with vertebrates. This may be due to the availability of a suitable response requirement. In this manuscript we offer a new method to explore operant conditioning in honey bees: the cap pushing response (CPR). We used the CPR to test for difference in learning curves between novel auto-shaping and more traditional explicit-shaping. The CPR protocol requires bees to exhibit a novel behavior by pushing a cap to uncover a food source. Using the CPR protocol we tested the effects of both explicit-shaping and auto-shaping techniques on operant conditioning. The goodness of fit and lack of fit of these data to the Rescorla-Wagner learning-curve model, widely used in classical conditioning studies, was tested. The model fit well to both control and explicit-shaping results, but only for a limited number of trials. Learning ceased rather than continuing to asymptotically approach the physiological most accurate possible. Rate of learning differed between shaped and control bee treatments. Learning rate was about 3 times faster for shaped bees, but for all measures of proficiency control and shaped bees reached the same level. Auto-shaped bees showed one-trial learning rather than the asymptotic approach to a maximal efficiency. However, in terms of return-time, the auto-shaped bees' learning did not carry over to the covered-well test treatments.

Squire et al. have proposed that trace and delay eyeblink conditioning procedures engage separate learning systems: a declarative hippocampal/cortical system associated with conscious contingency awareness, and a reflexive sub-cortical system independent of awareness, respectively (Clark and Squire, 1998; Smith et al., 2005). The only difference between these two procedures is that the conditioned stimulus (CS) and the unconditioned stimulus (US) overlap in delay conditioning, whereas there is a brief interval (e.g., 1s) between them in trace conditioning. In two experiments using the same procedure as Clark and Squire's group, we observed differential conditioning only in participants who showed contingency awareness in a post-experimental questionnaire, with both trace and delay procedures. We interpret these results to suggest that, although there may be multiple brain regions involved in learning, these regions are organized as a coordinated system rather than as separate, independent systems.

Investigations of fear conditioning in rodents and humans have illuminated the neural mechanisms underlying cued and contextual fear. A critical question is how personality dimensions such as trait anxiety act through these mechanisms to confer vulnerability to anxiety disorders, and whether humans' ability to overcome acquired fears depends on regulatory skills not characterized in animal models. In a neuroimaging study of fear conditioning in humans, we found evidence for two independent dimensions of neurocognitive function associated with trait vulnerability to anxiety. The first entailed increased amygdala responsivity to phasic fear cues. The second involved impoverished ventral prefrontal cortical (vPFC) recruitment to downregulate both cued and contextual fear prior to omission (extinction) of the aversive unconditioned stimulus. These two dimensions may contribute to symptomatology differences across anxiety disorders; the amygdala mechanism affecting the development of phobic fear and the frontal mechanism influencing the maintenance of both specific fears and generalized anxiety.

Influential theoretical accounts take the position that classical conditioning can induce placebo effects through conscious expectancies. In the current study two different conditioning procedures (hidden and open) were used to separate expectancy from conditioning in order to reveal the role of expectancy in the formation of nocebo hyperalgesia. Eighty-seven healthy females were randomly assigned to three groups (hidden conditioning, open conditioning, and control). Participants were selected according to the Fear of Pain Questionnaire scores and assigned to two subgroups: high and low level of fear of pain (trait). They received electrocutaneous pain stimuli preceded by either an orange or blue color. During the conditioning phase, one color was paired with pain stimuli of moderate intensity (control stimuli) and the other color was paired with pain stimuli of high intensity (nocebo stimuli) in both hidden and open conditioning groups. Only participants in the open conditioning group were informed about this association, however just before the testing phase the expectancy of hyperalgesia induced in this way was withdrawn. In the control group, both colors were followed by control pain stimuli. During the testing phase all participants received a series of stimuli of the same intensity, regardless of the preceding color. Participants rated pain intensity, expectancy of pain intensity and fear (state). We found that nocebo hyperalgesia was induced by hidden rather than open conditioning. The hidden conditioning procedure did not produce conscious expectancies related to pain. Nocebo hyperalgesia was induced in participants with low and high fear of pain and there was no difference in the magnitude of the nocebo effect between both groups. Nocebo hyperalgesia was not predicted by the fear of upcoming painful stimuli.

There is now ample evidence that the strength and underlying mechanisms of memory formation can be drastically altered by prior experience. However, the prior work using rodent models on this topic has used only males as subjects, and as a result, we do know whether or not the effects of prior experience on subsequent learning are similar in both sexes. As a first step towards addressing this shortcoming rats of both sexes were given auditory fear conditioning, or fear conditioning with unsignaled shocks, followed an hour or a day later by a single pairing of light and shock. Fear memory for each experience was assessed by measuring freezing behavior to the auditory cue and fear-potentiated startle to the light. Results showed that males trained with auditory fear conditioning showed facilitated learning to the subsequent visual fear conditioning session when the two training sessions were separated by one hour or one day. Females showed evidence of facilitation in rats given auditory conditioning when they were spaced by an hour, but not when they were spaced by one day. Contextual fear conditioning did not support the facilitation of subsequent learning under any conditions. These results indicate that the mechanism by which prior fear conditioning facilitates subsequent learning differs between sexes, and they set the stage for mechanistic studies to understand the neurobiological basis of this sex difference.

Neural plasticity in the amygdala is necessary for the acquisition and storage of memory in Pavlovian fear conditioning, but most neuroimaging studies have focused only on stimulus-evoked responses during the conditioning session. This study examined changes in the resting-state functional connectivity (RSFC) of the amygdala before and after Pavlovian fear conditioning, an emotional learning task. Behavioral results from the conditioning session revealed that participants learned normally and fMRI data recorded during learning identified a number of stimulus-evoked changes that were consistent with previous work. A direct comparison between the pre- and post-conditioning amygdala connectivity revealed a region of dorsal prefrontal cortex (PFC) in the superior frontal gyrus that showed a significant increase in connectivity following the conditioning session. A behavioral measure of explicit memory performance was positively correlated with the change in amygdala connectivity within a neighboring region in the superior frontal gyrus. Additionally, an implicit autonomic measure of conditioning was positively correlated with the change in connectivity between the amygdala and the anterior cingulate cortex (ACC). The resting-state data show that amygdala connectivity is altered following Pavlovian fear conditioning and that these changes are also related to behavioral outcomes. These alterations may reflect the operation of a consolidation process that strengthens neural connections to support memory after the learning event.

Prenatal cocaine exposure results in deficits in sensory preconditioning, discrimination reversal, and spatial navigation, tasks that require input from the hippocampus. However, there are no previous studies concerning prenatal cocaine effects on contextual fear conditioning, another hippocampal-dependent task. The present experiments tested whether chronic subcutaneous administration of 40 mg/kg of cocaine HCl to pregnant rats, from gestational day (GD) 8 through 20 would lead to disruption of contextual fear conditioning in adult male and female offspring. Offspring of saline-injected/pair-fed and untreated dams served as controls. Experiment 1 used a one-trial context conditioning preparation. Rats received a 2-s, 1-mA footshock in either the test context or a novel context, or received no shock on the day prior to the no-shock test. Defecation and freezing were measures of fear. Experiment 2 used a multiple measures protocol to optimize detection of prenatal treatment effects and was preceded by an open-field test. Rats received a 2-s, 0.8-mA footshock or no shock once daily over 4 days of conditioning. During 3 days of extinction, access to an adjacent chamber enabled the observation of four additional measures of fear: side crossing, latency, nose crossing, and side-differential. There were gender-dependent effects of conditioning on freezing and the four added measures of fear. Males showed higher levels of context conditioning and extinguished more slowly than females. The measures of nose crossing and side-differential revealed that prenatal cocaine exposure exaggerated gender-specific effects of context conditioning. The effects of prenatal cocaine exposure on context extinction are sexually dimorphic.

Nocebo hyperalgesia is a clinically relevant phenomenon and may be formed as a result of associative learning, implemented by classical conditioning. This study explored for the first time distinct nocebo conditioning methods and their consequences for nocebo attenuation methods. Healthy participants (N = 140) were recruited and randomized to the following nocebo hyperalgesia induction groups: conditioning with continuous reinforcement (CRF), conditioning with partial reinforcement (PRF), and a sham-conditioning control group. In the attenuation phase, counterconditioning was compared with extinction. During induction, participants experienced increased thermal pain in 100% of nocebo trials in the CRF groups, while in only 70% of nocebo trials in the PRF groups. During evocation, pain stimulation was equivalent across all trials. During attenuation, pain stimulation was decreased on nocebo trials relative to control trials for the counterconditioning groups, while pain remained equivalent across all trials for the extinction groups. Results showed that both PRF and CRF significantly induced nocebo hyperalgesia, but CRF was a more potent nocebo induction method, as compared to PRF. Counterconditioning was more effective than extinction in attenuating nocebo hyperalgesia. Neither CRF nor PRF resulted in resistance to extinction. However, compared with CRF, conditioning with PRF resulted in more resistance to counterconditioning. These findings demonstrate that the more ambiguous learning method of PRF can induce nocebo hyperalgesia and may potentially explain the treatment resistance and chronification seen in clinical practice. Further research is required to establish whether attenuation with counterconditioning is generalizable to clinical settings.

Contemporary computational accounts of instrumental conditioning have emphasized a role for a model-based system in which values are computed with reference to a rich model of the structure of the world, and a model-free system in which values are updated without encoding such structure. Much less studied is the possibility of a similar distinction operating at the level of Pavlovian conditioning. In the present study, we scanned human participants while they participated in a Pavlovian conditioning task with a simple structure while measuring activity in the human amygdala using a high-resolution fMRI protocol. After fitting a model-based algorithm and a variety of model-free algorithms to the fMRI data, we found evidence for the superiority of a model-based algorithm in accounting for activity in the amygdala compared to the model-free counterparts. These findings support an important role for model-based algorithms in describing the processes underpinning Pavlovian conditioning, as well as providing evidence of a role for the human amygdala in model-based inference.

Fear conditioning is an evolutionarily conserved type of learning serving as a model for the acquisition of situationally induced anxiety. Brain function supporting fear conditioning may be genetically influenced, which in part could explain genetic susceptibility for anxiety following stress exposure. Using a classical twin design and functional magnetic resonance imaging, we evaluated genetic influences (h2) on brain activity and standard autonomic measures during fear conditioning. We found an additive genetic influence on mean brain activation (h2 = 0.34) and autonomic responses (h2 = 0.24) during fear learning. The experiment also allowed estimation of the genetic influence on brain activation during safety learning (h2 = 0.55). The mean safety, but not fear, related brain activation was genetically correlated with autonomic responses. We conclude that fear and safety learning processes, both involved in anxiety development, are moderately genetically influenced as expressed both in the brain and the body.

Consumption of a high concentration of sucrose can have either a detrimental, negative contrast effect or a facilitatory, preference conditioning effect on subsequent consumption of a low concentration of sucrose, depending on the cues that are present during consumption. The role of context and flavor cues in determining these effects were studied using analysis of the microstructure of licking in mice. Exposure to a high concentration followed by exposure to a low concentration resulted in a transient reduction in mean lick cluster size, which was context dependent (Experiment 1). However, there was no change in the total number of licks or overall consumption. When a flavor that had previously been paired with a high concentration was paired with a low concentration, there was an increase in the total number of licks, and overall consumption, but no change in the mean lick cluster size (Experiment 2). Pairing a high concentration with a flavor in a particular context before pairing the context and flavor compound with a low concentration resulted in abolishing the expression of the flavor preference conditioning effect on the total number of licks and consumption (Experiment 3). These results demonstrate that although context and flavor cues have dissociable effects on licking behavior, their interaction has an antagonistic effect on the behavioral expression of memory.

Pavlovian fear conditioning is an evolutionary conserved and extensively studied form of associative learning and memory. In mammals, the lateral amygdala (LA) is an essential locus for Pavlovian fear learning and memory. Despite significant progress unraveling the cellular mechanisms responsible for fear conditioning, very little is known about the anatomical organization of neurons encoding fear conditioning in the LA. One key question is how fear conditioning to different sensory stimuli is organized in LA neuronal ensembles. Here we show that Pavlovian fear conditioning, formed through either the auditory or visual sensory modality, activates a similar density of LA neurons expressing a learning-induced phosphorylated extracellular signal-regulated kinase (p-ERK1/2). While the size of the neuron population specific to either memory was similar, the anatomical distribution differed. Several discrete sites in the LA contained a small but significant number of p-ERK1/2-expressing neurons specific to either sensory modality. The sites were anatomically localized to different levels of the longitudinal plane and were independent of both memory strength and the relative size of the activated neuronal population, suggesting some portion of the memory trace for auditory and visually cued fear conditioning is allocated differently in the LA. Presenting the visual stimulus by itself did not activate the same p-ERK1/2 neuron density or pattern, confirming the novelty of light alone cannot account for the specific pattern of activated neurons after visual fear conditioning. Together, these findings reveal an anatomical distribution of visual and auditory fear conditioning at the level of neuronal ensembles in the LA.

New learning results in modulation of intrinsic plasticity in the underlying brain regions. Such changes in intrinsic plasticity can influence allocation and encoding of future memories such that new memories encoded during the period of enhanced excitability are linked to the original memory. The temporal window during which the two memories interact depends upon the time course of intrinsic plasticity following new learning.

Several studies have reported regulatory effects of estrogens on fear conditioning in female rodents. However, these studies used different doses, durations, and/or administration methods, and reported inconsistent results. To clarify the effect of estrogen on fear conditioning, we investigated the effects of different doses and durations of estradiol administration on freezing behavior during contextual fear conditioning in ovariectomized (OVX) mice. In Experiment 1, OVX ICR mice received a single subcutaneous (s.c.) injection of either oil vehicle (control, 0.1 ml sesame oil) or varied doses (0.5 μg/0.1 ml, 5 μg/0.1 ml, or 50 μg/0.1 ml) of 17β-estradiol-3-benzoate (EB). Fear conditioning was conducted two days post-EB treatment, and the mice were tested for the learned fear response the following day. In Experiment 2, OVX female mice received an s.c. implantation of a Silastic capsule (I.D. 1.98 × 20.0 mm) containing either vehicle or varied doses (0.05 μg/0.1 ml, 0.5 μg/0.1 ml, 5 μg/0.1 ml, 50 μg/0.1 ml) of EB. Two weeks after implantation, fear conditioning was conducted. During the tests conducted 24 h after conditioning, the high dose EB group showed longer freezing times in both experiments, and lower locomotor activity compared to the control or lower dose groups. In Experiment 3, serum estradiol concentrations of the mice that were treated like those in Experiment 2, were measured; the serum levels of estradiol increased linearly according to the dose of EB administered. The results suggest that mice treated with a high dose of EB exhibit enhanced fear learning, regardless of treatment duration. As a woman's vulnerability to emotional disorders increases in the peripregnancy period, during which estrogen levels are high, the results from the high-dose EB groups may be important for understanding the hormonal mechanisms involved in these disorders.

A classical conditioning framework is often used for clinical reasoning about pain that persists after tissue healing. However, experimental studies demonstrating classically conditioned pain in humans are lacking. The current study tested whether non-nociceptive somatosensory stimuli can come to modulate pain thresholds after being paired with painful nociceptive stimuli in healthy humans. We used a differential simultaneous conditioning paradigm in which one nonpainful vibrotactile conditioned stimulus (CS(+)) was simultaneously paired with an unconditioned painful laser stimulus, and another vibrotactile stimulus (CS(-)) was paired with a nonpainful laser stimulus. After acquisition, at-pain-threshold laser stimuli were delivered simultaneously with a CS(+) or CS(-) vibrotactile stimulus. The primary outcome was the percentage of at-threshold laser stimuli that were reported as painful. The results were as expected: after conditioning, at-threshold laser trials paired with the CS(+) were reported as painful more often, as more intense, and as more unpleasant than those paired with the CS(-). This study provides new evidence that pain thresholds can be modulated via classical conditioning, even when the stimulus used to test the threshold cannot be anticipated. As such, it lays a critical foundation for further investigations of classical conditioning as a possible driver of persistent pain.

Memory consolidation requires transcription and translation of new protein. Arc, an effector immediate early gene, and zif268, a regulatory transcription factor, have been implicated in synaptic plasticity underlying learning and memory. This study explored the temporal expression profiles of these proteins in the rat hippocampus following fear conditioning. We observed a time-dependent increase of Arc protein in the dorsal hippocampus 30-to-90-minute post training, returning to basal levels at 4 h. Zif268 protein levels, however, gradually increased at 30-minute post training before peaking in expression at 60 minute. The timing of hippocampal Arc and zif268 expression coincides with the critical period for protein synthesis-dependent memory consolidation following fear conditioning. However, the expression of Arc protein appears to be driven by context exploration, whereas, zif268 expression may be more specifically related to associative learning. These findings suggest that altered Arc and zif268 expression are related to neural plasticity during the formation of fear memory.

Repeated exposure to threatening stimuli alters sensory responses. We investigated the underlying neural mechanism by re-analyzing previously published simultaneous electroencephalogram-functional magnetic resonance imaging (EEG-fMRI) data from humans viewing oriented gratings during Pavlovian fear conditioning. In acquisition, one grating (CS+) was paired with a noxious noise, the unconditioned stimulus (US). The other grating (CS-) was never paired with the US. In habituation, which preceded acquisition, and in extinction, the same two gratings were presented without US. Using fMRI multivoxel patterns in primary visual cortex during habituation as reference, we found that during acquisition, aversive learning selectively prompted systematic changes in multivoxel patterns evoked by CS+. Specifically, CS+ evoked voxel patterns in V1 became sparser as aversive learning progressed, and the sparsified pattern appeared to be preserved in extinction. Concomitant with the voxel pattern changes, occipital alpha oscillations were increasingly more desynchronized during CS+ (but not CS-) trials. Across acquisition trials, the rate of change in CS+-related alpha desynchronization was correlated with the rate of change in multivoxel pattern representations of CS+. Furthermore, alpha oscillations co-varied with blood-oxygen-level-dependent (BOLD) data in the ventral attention network, but not with BOLD in the amygdala. Thus, fear conditioning prompts persistent sparsification of voxel patterns evoked by threat, likely mediated by attention-related mechanisms.