Fear conditioning and extinction: influence of sex and menstrual cycle in healthy humans

Although sex differences have been demonstrated in behavioral paradigms of fear conditioning, the findings have been inconsistent, and fear extinction has been little studied. The present study investigated the influence of sex and menstrual cycle phase on the recall of fear extinction. Three groups of healthy adult participants were studied: women at 2 different phases of the menstrual cycle (early follicular [early cycle] and late follicular [midcycle]) and men. Participants underwent a 2-day fear conditioning and extinction protocol. The paradigm entailed habituation, fear conditioning, and extinction learning on Day 1 and extinction recall and fear renewal on Day 2. Skin conductance served as the dependent variable. During fear acquisition on Day 1, men showed significantly larger conditioned responses relative to women; early cycle and midcycle women did not differ. No significant group differences were found during extinction learning. On Day 2, men and early cycle women expressed greater extinction memory than midcycle women. These data confirm sex differences in conditioned fear acquisition and suggest that midcycle hormones attenuate extinction recall.     

Estrous cycle phase and gonadal hormones influence conditioned fear extinction

Gonadal hormones modulate fear acquisition, but less is known about the influence of gonadal hormones on fear extinction. We assessed sex differences and the influence of gonadal hormone fluctuations and exogenous manipulations of estrogen and progesterone on acquisition, extinction learning and extinction recall in a 3 day auditory fear conditioning and extinction protocol. Experiments were conducted on males and naturally cycling female rats. Regarding female rats, significant differences in fear extinction were observed between subgroups of females, depending on their phase of the estrous cycle. Extinction that took place during the proestrus (high estrogen/progesterone) phase was more fully consolidated, as evidenced by low freezing during a recall test. This suggests that estrogen and/or progesterone facilitate extinction. In support of this, injection of both estrogen and progesterone prior to extinction learning in female rats during the metestrus phase of the cycle (low estrogen/progesterone) facilitated extinction consolidation, and blockade of estrogen and progesterone receptors during the proestrus phase impaired extinction consolidation. When comparing male to female rats without consideration of the estrous cycle phase, no significant sex differences were observed. When accounting for cycle phase in females, sex differences were observed only during extinction recall. Female rats that underwent extinction during the metestrus phase showed significantly higher freezing during the recall test relative to males. Collectively, these data suggest that gonadal hormones influence extinction behavior possibly by influencing the function of brain regions involved in the consolidation of fear extinction. Moreover, the elevated fear observed in female relative to male rats during extinction recall suggests that gonadal hormones may in part play a role in the higher prevalence of anxiety disorders in women.     

Measuring the conditioned response: A comparison of pupillometry,skin conductance,and startle electromyography

In human fear conditioning studies, different physiological readouts can be used to track conditioned responding during fear learning. Commonly employed readouts such as skin conductance responses (SCR) or startle responses have in recent years been complemented by pupillary readouts, but to date it is unknown how pupillary readouts relate to other measures of the conditioned response. To examine differences and communalities among pupil responses, SCR, and startle responses, we simultaneously recorded pupil diameter, skin conductance, and startle electromyography in 47 healthy subjects during fear acquisition, extinction, and a recall test on 2 consecutive days. The different measures correlated only weakly, displaying most prominent differences in their response patterns during fear acquisition. Whereas SCR and startle responses habituated, pupillary measures did not. Instead, they increased in response to fear conditioned stimuli and most closely followed ratings of unconditioned stimulus (US) expectancy. Moreover, we observed that startle‐induced pupil responses showed stimulus discrimination during fear acquisition, suggesting a fear potentiation of the auditory pupil reflex. We conclude that different physiological outcome measures of the conditioned response inform about different cognitive‐affective processes during fear learning, with pupil responses being least affected by physiological habituation and most closely following US expectancy.     

Brain morphology correlates of interindividual differences in conditioned fear acquisition and extinction learning

The neural circuits underlying fear learning have been intensively investigated in pavlovian fear conditioning paradigms across species. These studies established a predominant role for the amygdala in fear acquisition, while the ventromedial prefrontal cortex (vmPFC) has been shown to be important in the extinction of conditioned fear. However, studies on morphological correlates of fear learning could not consistently confirm an association with these structures. The objective of the present study was to investigate if interindividual differences in morphology of the amygdala and the vmPFC are related to differences in fear acquisition and extinction learning in humans. We performed structural magnetic resonance imaging in 68 healthy participants who underwent a differential cued fear conditioning paradigm. Volumes of subcortical structures as well as cortical thickness were computed by the semi-automated segmentation software Freesurfer. Stronger acquisition of fear as indexed by skin conductance responses was associated with larger right amygdala volume, while the degree of extinction learning was positively correlated with cortical thickness of the right vmPFC. Both findings could be conceptually replicated in an independent sample of 53 subjects. The data complement our understanding of the role of human brain morphology in the mechanisms of the acquisition and extinction of conditioned fear.     

Conditioned and extinguished fear modulate functional corticocardiac coupling in humans

Although the conditioned cardiac fear response is an important index of psychophysiological fear processing, underlying neural mechanisms remain unclear. N = 22 participants underwent differential fear conditioning and extinction with face pictures as conditioned stimuli (CS) and loud noise bursts as aversive unconditioned stimulus (US) on Day 1 and a recall test 1 day later. We assessed ERPs, evoked heart period (HP), and time‐lagged within‐subject correlations of single‐trial EEG amplitude and HP as index for corticocardiac coupling in response to the CS. Fear‐conditioned stimuli (CS+) triggered cardiac deceleration during fear acquisition and recall. Meanwhile, only during Day 1 acquisition, CS+ evoked larger late positivities in the ERP than CS?. Most importantly, during Day 2 recall, stimulus‐evoked single‐trial EEG responses in the time window between 250 and 500 ms predicted the magnitude of cardiac fear responses 2 to 5 s later. This marker of corticocardiac coupling selectively emerged in response to not previously extinguished CS+ but was absent in response to CS? or previously extinguished CS+. The present results provide first evidence that fear conditioning and extinction modulate functional corticocardiac coupling in humans. Underlying mechanisms may involve subcortical structures enhancing corticocardiac transmission to facilitate processing of consolidated conditioned fear.     

Effects of sex, phase of the menstrual cycle and gonadal hormones on pain in healthy humans

Sex differences in pain have been noted; women typically report more pain than men. Gonadal hormones may influence pain reports, and, moreover, such hormones may help to explain sex differences and menstrual cycle differences in pain. This study measured venipuncture and intravenous catherization pain during the follicular and luteal phases of the menstrual cycle in regularly menstruating women. Pain was also assessed in a group of men. Pain ratings were higher in women than men. In women, pain ratings did not differ between the follicular and luteal phases. Estradiol and progesterone increased from follicular to luteal phases. Within-phase analyses revealed that pain ratings were positively correlated with estradiol and progesterone during the luteal phase. Moreover, increases in estradiol and progesterone across the menstrual cycle were positively correlated with increases in pain. These findings suggest that variations in gonadal hormones during the menstrual cycle influence the experience of pain in healthy women.     

Reversible inactivations of the cerebellum with muscimol prevent the acquisition and extinction of conditioned nictitating membrane responses in the rabbit

Lesions of the cerebellum severely impair the classically conditioned nictitating membrane response (NMR) in rabbits. Thus, the cerebellum is essential for the production of conditioned responses (CRs), either because it is actively involved in NMR conditioning or because damage to it causes motor or other general deficits. To distinguish between these alternatives, the cerebellum may be inactivated during training. Inactivation of the cerebellum during acquisition training might result in the absence of CRs on initial trials of subsequent training without the neuronal blockade. The blockade may have prevented learning but it may have produced other deficits that require time or further training to overcome. This problem can be addressed by inactivating the cerebellum during extinction training. If inactivation during extinction training results in the immediate production of CRs when training is resumed without the blockade, then it may be concluded that extinction learning was prevented by the blockade — the presence of CRs argues against any deficits not associated with learning. We used muscimol to inactivate the cerebellum and test its involvement in acquisition and extinction of NMR conditioning in the same subjects. We injected muscimol close to the interpositus nucleus of the cerebellum 1 h before each of four daily training sessions of delay conditioning. Almost no CRs were produced in these training sessions — there was little or no acquisition of NMR conditioning during cerebellar inactivation. The subjects were then trained for four daily sessions without injections of muscimol. There were no CRs on initial trials of the first session of retraining, but all subjects produced CRs by the end of this session. The subjects then received four daily sessions of extinction training with muscimol inactivation of the nuclei — no CRs were produced. Extinction training then continued for four daily sessions without muscimol inactivation. On the first of these sessions, all subjects immediately produced high levels of CRs. These responses then extinguished within and between sessions with characteristic beginning-of-session spontaneous recovery. There was little or no extinction of NMR conditioning during cerebellar inactivation. After inactivation, the muscimolinactivated subjects went on to acquire and extinguish NM responses at rates similar to those of appropriate controls. We conclude that cerebellar circuitry is essential for, and actively engaged in, both acquisition and extinction of this simple form of motor learning.     

Positron emission tomographic imaging of neural correlates of a fear acquisition and extinction paradigm in women with childhood sexual-abuse-related post-traumatic stress disorder

BACKGROUND: In the conditioned fear paradigm, repeated pairing of an aversive unconditioned stimulus (US) (e.g. electric shock) with a neutral conditioned stimulus (CS) (e.g. bright light) results in a conditioned fear response to the light alone. Animal studies have shown that the amygdala plays a critical role in acquisition of conditioned fear responses, while the medial prefrontal cortex (including anterior cingulate), through inhibition of amygdala responsiveness, has been hypothesized to play a role in extinction of fear responses. No studies have examined neural correlates of fear conditioning and extinction in patients with post-traumatic stress disorder (PTSD). METHOD: Women with early childhood sexual-abuse-related PTSD (n = 8) and women without abuse or PTSD (n = 11) underwent measurement of psychophysiological (skin conductance) responding as well as positron emission tomographic (PET) measurement of cerebral blood flow during habituation, acquisition and extinction conditions. During habituation subjects were repeatedly exposed to a blue square on a screen. During acquisition, exposure to the blue square (CS) was paired with an electric shock to the forearm (US). With extinction, subjects were again exposed to the blue squares without shock. On a different day subjects went through the same procedure with electric shocks administered randomly in the absence of the blue square. RESULTS: Skin conductance responding to the CS was consistent with the development of conditioned responses with this paradigm. PTSD patients had increased left amygdala activation with fear acquisition, and decreased anterior cingulate function during extinction, relative to controls. CONCLUSIONS: These findings implicate amygdala and anterior cingulate in the acquisition and extinction of fear responses, respectively, in PTSD.     

Fear extinction in humans: Effects of acquisition-extinction delay and masked stimulus presentations

Fear extinction can be viewed as an inhibitory learning process. This is supported by post-extinction phenomena demonstrating the return of fear, such as reinstatement. Recent work has questioned this account, claiming that extinction initiated immediately after fear acquisition can abolish the return of fear. In the current study, participants were fear conditioned to four different conditioned stimuli (CS) and underwent extinction either immediately or after a 24h delay. During extinction, we manipulated CS contingency awareness by presenting two of the CSs (one CS+, one CS-) under non-masked conditions and the other two CSs under masked conditions. Compared to delayed extinction, immediate extinction of non-masked CSs promoted less extinction of fear-potentiated startle and shock expectancy ratings and less reinstatement of fear-potentiated startle without affecting shock expectancy ratings. Critically, future research should clarify how the differences between immediate and delayed extinction in within-session extinction modulate the recovery of fear.     

Electrodermal conditioning to potentially phobic stimuli in male and female subjects

There are clear sex differences in incidence of phobias for small animals, and in questionnaire-measured fear for animals. The present study examined whether these sex differences were reflected also in electrodermal conditioning to potentially phobic stimuli. Separate groups of males and females were exposed to a conditioning session involving either potentially phobic, snakes and spiders, or fear-irrelevant, flowers and mushrooms, conditioned stimuli with electric shock as the unconditioned stimulus. A long interstimulus interval differential paradigm was used, allowing analysis of first- and second interval anticipatory responses, and third-interval omission responses. There were 8 habituation trials, 16 acquisition trials and 40 extinction trials. Half of the trials involved the reinforced cue, and the other half the unreinforced cue. There were clear conditioning effects, with superior acquisition and resistance to extinction to the phobic as compared to the fear-irrelevant stimuli. However, there were no differences between the two sexes. The results were interpreted in terms of the preparedness theory of phobias, and in terms of social learning factors.     

Sex differences in verbal learning

Although sex differences in cognitive abilities have been reported in the experimental literature, there has been a paucity of information about how men and women might perform differently on clinical neuropsychological tests. The present study examines sex differences in verbal learning. Sixtyeight men and 68 women of equal age and education were administered the California Verbal Learning Test. Dependent variables included measures of recall, recognition, learning characteristics, and error types. Women displayed consistently higher levels of immedite and delayed free recall and made greater use of semantic clustering. There were no sex differences on recognition testing or error types. Results suggest that women's superior recall is attributable to better retrieval, which, in turn, is related to their greater use of verbally mediated strategies. This study highlights the importance of investigating sex differences on clinical instruments and reporting separate sex norms when appropriate.     

Stress hormones are associated with the neuronal correlates of instructed fear conditioning

The effects of sex and stress hormones on classical fear conditioning have been subject of recent experimental studies. A correlation approach between basal cortisol concentrations and neuronal activation in fear-related structures seems to be a promising alternative approach in order to foster our understanding of how cortisol influences emotional learning. In this functional magnetic resonance imaging study, participants with varying sex hormone status (20 men, 15 women taking oral contraceptives, 15 women tested in the luteal phase) underwent an instructed fear conditioning protocol with geometrical figures as conditioned stimuli and an electrical stimulation as unconditioned stimulus. Salivary cortisol concentrations were measured and afterwards correlated with fear conditioned brain responses. Results revealed a positive correlation between basal cortisol levels and differential activation in the amygdala in men and OC women only. These results suggest that elevated endogenous cortisol levels are associated with enhanced fear anticipation depending on current sex hormone availability.     

Amphetamine and extinction of cued fear

Much research is focused on developing novel drugs to improve memory. In particular, psychostimulants have been shown to enhance memory and have a long history of safe use in humans. In prior work, we have shown that very low doses of amphetamine administered before training on a Pavlovian fear-conditioning task can dramatically facilitate the acquisition of cued fear. The current experiment sought to expand these findings to the extinction of cued fear, a well-known paradigm with therapeutic implications for learned phobias and post-traumatic stress disorder. If extinction reflects new learning, one might expect drugs that enhance the acquisition of cued fear to also enhance the extinction of cued fear. This experiment examined whether 0.005 or 0.05 mg/kg of d-amphetamine (therapeutic doses shown to enhance acquisition) also enhance the extinction of cued fear. Contrary to our hypothesis, amphetamine did not accelerate extinction. Thus, at doses that enhance acquisition of conditioned fear, amphetamine does not appear to enhance extinction.     

Network model of fear extinction and renewal functional pathways

The objective of this study was to examine the opposite behavior responses of conditioned fear extinction and renewal and how they are represented by network interactions between brain regions. This work is a continuation of a series of brain mapping studies of various inhibitory phenomena, including conditioned inhibition, blocking and extinction. A tone-footshock fear conditioning paradigm in rats was used, followed by extinction and testing in two different contexts. Fluorodeoxyglucose autoradiography was used to compare mean regional brain activity and interregional correlations resulting from the presentation of the extinguished tone in or out of the extinction context. A confirmatory structural equation model, constructed from a neural network proposed to underlie fear extinction, showed a reversal from negative regional interactions during extinction recall to positive interactions during fear renewal. Additionally, the magnitude of direct effects was different between groups, reflecting a change in the strength of the influences conveyed through those pathways. The results suggest that the extinguished tone encountered outside of the extinction context recruits auditory and limbic areas, which in turn influence the interactions of the infralimbic cortex with the amygdala and ventrolateral periaqueductal gray. Interestingly, the results also suggest that two independent pathways influence conditioned freezing: one from the central amygdaloid nucleus and the other from the infralimbic cortex directly to the ventrolateral periaqueductal gray.     

Timing of extinction relative to acquisition: a parametric analysis of fear extinction in humans

Fear extinction is a reduction in conditioned fear following repeated exposure to the feared cue in the absence of any aversive event. Extinguished fear often reappears after extinction through spontaneous recovery. Animal studies suggest that spontaneous recovery can be abolished if extinction occurs within minutes of acquisition. However, a limited number of human extinction studies have shown that short interval extinction does not prevent the return of fear. For this reason, we performed an in-depth parametric analysis of human fear extinction using fear-potentiated startle. Using separate single-cue and differential conditioning paradigms, participants were fear conditioned and then underwent extinction either 10 min (Immediate) or 72 hr (Delayed) later. Testing for spontaneous recovery occurred 96 hr after acquisition. In the single cue paradigm, the Immediate and Delayed groups exhibited differences in context, but not fear, conditioning. With differential conditioning, there were no differences in context conditioning and the Immediate group displayed less spontaneous recovery. Thus, the results remain inconclusive regarding spontaneous recovery and the timing of extinction and are discussed in terms of performing translational studies of fear in humans.     

Carbachol injections into the nucleus accumbens disrupt acquisition and expression of fear-potentiated startle and freezing in rats

The nucleus accumbens is involved in different types of emotional learning, ranging from appetitive instrumental learning to Pavlovian fear conditioning. In previous studies, we found that temporary inactivation of the nucleus accumbens blocked both the acquisition and expression of conditioned fear. This was not due to altered dopaminergic activity as we have also found that intra-nucleus accumbens infusions of the dopamine agonist amphetamine do not affect either the acquisition or the expression of conditioned fear. Therefore, in the present study we examined whether cholinergic activity in the nucleus accumbens is involved in the acquisition and expression of conditioned fear. Specifically, the effect of intra-nucleus accumbens infusions of the unselective cholinergic agonist carbachol on the acquisition and expression of conditioned fear was assessed. Across several experiments, we measured fear to visual and acoustic conditioned stimuli and to the experimental context. Further, two different measures of conditioned fear were recorded: fear potentiation of startle and freezing. Intra-nucleus accumbens carbachol infusions disrupted acquisition as well as expression of conditioned fear, regardless of the modality of the fear-eliciting stimulus or of the specific measure of conditioned fear. This disruption of conditioned fear was not simply a by-product of enhanced motor activity which also occurred after intra-nucleus accumbens carbachol infusions. Interestingly, despite the substantial effect of intra-nucleus accumbens carbachol on expression of conditioned fear, the results of the final experiment suggest that these rats extinguish similarly to control rats. Taken together, the present results indicate that acetylcholine within the nucleus accumbens is important for the learning and retrieval of conditioned fear.     

Sex difference in rabbit eyelid conditioning

The rabbit eyelid conditioned reflex has been used to compare associative learning in males and ovariectomized females. A new method for monitoring eyelid movements is described. Rabbits were trained on simple delay classical conditioning. Conditioned responses were recorded during 8 acquisition days and 6 days of extinction training. Analysis of variance (ANOVA) and the least significant difference (LSD) post hoc test was used to analyze the data. The results showed that males achieved significantly better learning than females during the first day of acquisition but later they slowly attained the best result, contrary to females. Moreover, extinction of the conditioned reflex was significantly faster in females than in males. It is postulated that females learn and extinguish faster than males because of a higher level of brain plasticity.     

Differential involvement of medial prefrontal cortex and basolateral amygdala extracellular signal-regulated kinase in extinction of conditioned taste aversion is dependent on different intervals of extinction following conditioning

Extinction reflects a decrease in the conditioned response (CR) following non-reinforcement of a conditioned stimulus. Behavioral evidence indicates that extinction involves an inhibitory learning mechanism in which the extinguished CR reappears with presentation of an unconditioned stimulus. However, recent studies on fear conditioning suggest that extinction erases the original conditioning if the time interval between fear acquisition and extinction is short. The present study examined the effects of different intervals between acquisition and extinction of the original memory in conditioned taste aversion (CTA). Male Long-Evans rats acquired CTA by associating a 0.2% sucrose solution with malaise induced by i.p. injection of 4 ml/kg 0.15 M LiCl. Two different time intervals, 5 and 24 h, between CTA acquisition and extinction were used. Five or 24 h after CTA acquisition, extinction trials were performed, in which a bottle containing 20 ml of a 0.2% sucrose solution was provided for 10 min without subsequent LiCl injection. If sucrose consumption during the extinction trials was greater than the average water consumption, then rats were considered to have reached CTA extinction. Rats subjected to extinction trials lasting 24 h, but not 5 h, after acquisition re-exhibited the extinguished CR following injection of 0.15 M LiCl alone 7 days after acquisition. Extracellular signal-regulated kinase (ERK) in the medial prefrontal cortex (mPFC) and basolateral nucleus of the amygdala (BLA) was examined by Western blot after the first extinction trial. ERK activation in the mPFC was induced after the extinction trial beginning 5 h after acquisition, whereas the extinction trial performed 24 h after acquisition induced ERK activation in the BLA. These data suggest that the original conditioning can be inhibited or retained by CTA extinction depending on the time interval between acquisition and extinction and that the ERK transduction pathway in the mPFC and BLA is differentially involved in these processes.     

Sex differences in learning processes of classical and operant conditioning

Males and females learn and remember differently at different times in their lives. These differences occur in most species, from invertebrates to humans. We review here sex differences as they occur in laboratory rodent species. We focus on classical and operant conditioning paradigms, including classical eyeblink conditioning, fear-conditioning, active avoidance and conditioned taste aversion. Sex differences have been reported during acquisition, retention and extinction in most of these paradigms. In general, females perform better than males in the classical eyeblink conditioning, in fear-potentiated startle and in most operant conditioning tasks, such as the active avoidance test. However, in the classical fear-conditioning paradigm, in certain lever-pressing paradigms and in the conditioned taste aversion, males outperform females or are more resistant to extinction. Most sex differences in conditioning are dependent on organizational effects of gonadal hormones during early development of the brain, in addition to modulation by activational effects during puberty and adulthood. Critically, sex differences in performance account for some of the reported effects on learning and these are discussed throughout the review. Because so many mental disorders are more prevalent in one sex than the other, it is important to consider sex differences in learning when applying animal models of learning for these disorders. Finally, we discuss how sex differences in learning continue to alter the brain throughout the lifespan. Thus, sex differences in learning are not only mediated by sex differences in the brain, but also contribute to them.     

Context modulation of memory for fear extinction in humans

Distinct memories are formed during fear conditioning and subsequent extinction. In animals, the expression of the latter is gated by the context. The recall of extinction memory after a long delay, and the contextual modulation thereof, has not been directly tested in humans. Mentally healthy volunteers underwent a 2-day fear conditioning and extinction protocol that examined the recall of extinction memory and its relationship to context. Conditioned stimuli were paired with an aversive electric shock in one visual context and extinguished in a different context. Extinction recall and renewal were examined 24 h after training. We found that skin conductance responses were small when the conditioned stimulus was presented in the extinction context, but responses were renewed when the conditioned stimulus was presented in the conditioning context. This finding demonstrates context dependency of extinction recall in humans.