The influence of gonadal hormones on conditioned fear extinction in healthy humans

Recent rodent studies suggest that gonadal hormones influence extinction of conditioned fear. Here we investigated sex differences in, and the influence of estradiol and progesterone on, fear extinction in healthy humans. Men and women underwent a two-day paradigm in which fear conditioning and extinction learning took place on day 1 and extinction recall was tested on day 2. Visual cues were used as the conditioned stimuli and a mild electric shock was used as the unconditioned stimulus. Skin conductance was recorded throughout the experiment and used to measure conditioned responses (CRs). Blood samples were obtained from all women to measure estradiol and progesterone levels. We found that higher estradiol during extinction learning enhanced subsequent extinction recall but had no effects on fear acquisition or extinction learning itself. Sex differences were only observed during acquisition, with men exhibiting significantly higher CRs. After dividing women into low- and high-estradiol groups, men showed comparable extinction recall to high-estradiol women, and both of these groups showed higher extinction recall than low-estradiol women. Therefore, sex differences in extinction memory emerged only after taking into account women's estradiol levels. Lower estradiol may impair extinction consolidation in women. These findings could have practical applications in the treatment of anxiety disorders through cognitive and behavioral therapies.     

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.     

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.     

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.     

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.     

Human fear conditioning and extinction: Timing is everything…or is it?

A differential fear conditioning paradigm was used with 107 healthy undergraduate participants to evaluate the effect of conditioned stimulus (CS) temporal properties on fear acquisition and extinction. Two minute duration CSs were used for Day 1 fear acquisition. Participants were randomized to receive either 1, 2, or 4 min CS durations during Day 2 extinction. Extinction re-test was examined on Day 3 using the original acquisition CS duration (2 min). Findings indicated that participants who were aware of the CS+/unconditioned stimulus (US) contingency (n = 52) develop a temporal expectation about when the unconditioned stimulus will be delivered. Although the shorter duration CS resulted in greater fear reduction during extinction, cessation of fear responding at re-test was the same for CS extinction durations ranging from half the CS acquisition duration to twice the CS acquisition duration. Thus, extinction performance did not predict extinction at re-test, which could have important implications for optimizing exposure therapy for anxiety disorders.     

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.     

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.     

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.     

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.     

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.     

Instructed extinction differentially affects the emotional and cognitive expression of associative fear memory

Instructed extinction after fear conditioning is relatively effective in attenuating electrodermal responding. Testing the single‐process account of fear learning, we examined whether this manipulation similarly affects the startle response. Skin conductance responses (SCRs), startle responses, and online unconditioned stimulus (US) expectancy ratings were measured during fear acquisition (Day 1), extinction, and reinstatement (Day 2). Before extinction onset, half of the subjects were instructed that the conditioned stimulus would not be followed by the US (Instructed Extinction) whereas the other subjects were not instructed (Normal Extinction). This simple instruction completely abolished both differential SCR and US expectancy ratings, but not the startle fear response. Although the manipulation facilitated extinction learning, it did not prevent the recovery of the startle response. The present findings are better explained by a dual‐ rather than a single‐process account of fear learning.     

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.     

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.     

Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state

The heterogeneity of follicle stimulating hormone (FSH) and luteinizing hormone (LH) was investigated in five women aged 29.4 +/- 3.2 years (mean +/- SD) throughout their menstrual cycles and in five post- menopausal women aged 53.8 +/- 5.6 years. Chromatofocusing (pH range 7- 4) revealed menstrual cycle stage- and postmenopausal-related differences in the serum gonadotrophin charge. There were differences in the proportion of FSH with an isoelectric point (pl) > 4.3 across phases of the menstrual cycle (P = 0.019): midcycle (MC) 50%; early to mid-follicular (EMF) 36%; late follicular (LF) 37%, luteal (L) 29% and following the menopause (PM) 17%. There was no significant difference in the proportion of LH with pl > 6.55 between midcycle (53%) and EMF, LF or L phases (36, 43 and 32% respectively); although all were greater than that found in the menopause (13%). Concanavalin A chromatography revealed less (P < 0.005) complex FSH and LH glycoforms at midcycle (63 and 13%) than in the EMF, LF and L phases (90 and 18; 90 and 20 and 93 and 24% respectively). Menopausal gonadotrophins were least complex (FSH 34%, LH 4%). There was a direct relationship between serum FSH and FSH pl/complexity, and less acidic FSH was associated with reduced FSH complexity. Increased oestradiol was associated with basic FSH isoforms during the menstrual cycle and reduced follicular phase FSH complexity. We conclude that changes in gonadotrophin glycoforms occur through the menstrual cycle which are related to changes in the prevailing steroid environment. Following the menopause oestrogenic loss resulted in acidic, relatively simple glycoforms.      

Influence of endogenous and exogenous sex hormones on plasma brain-derived neurotrophic factor

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a mediator of neuronal plasticity and influences learning, memory and cognitive behaviour. The aim of this study is to assess plasma BDNF variations according to hormonal status. METHODS: A total of 60 subjects were included: 20 fertile ovulatory women, 15 amenorrhoeic women and 25 postmenopausal women. Blood samples were collected after overnight fasting. For 5 out of the 20 fertile women, samples were collected every 2 days throughout the whole menstrual cycle. Following basal evaluation, 10 out of 25 postmenopausal women were administered a hormone replacement therapy (HRT) and reevaluated after 6 months of treatment. Plasma BDNF concentrations were measured by enzyme-linked immunosorbent assay. In fertile women, estradiol (E(2)), progesterone and gonadotrophins were also assessed. RESULTS: In fertile women, luteal phase levels of plasma BDNF were significantly higher than follicular phase levels (P < 0.001). BDNF increased from early follicular phase up to Day 14 of the cycle, reaching a pre-ovulatory peak, similar to E(2). A second rise took place during mid-luteal phase, with a peak on Day 24. Amenorrhoeic subjects, as well as postmenopausal women, showed significantly lower plasma BDNF levels compared with fertile females (P < 0.001). BDNF was positively correlated with E(2) and progesterone and negatively correlated with menopausal age. HRT restored BDNF levels to those present in fertile women during the follicular phase. CONCLUSIONS: Plasma BDNF levels are influenced by hormonal status. Modifications in BDNF circulating levels during the menstrual cycle suggest a potential role for gonadal sex hormones (E(2) and progesterone) in regulating neurotrophin expression.     

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.     

The effects of neurotoxic hippocampal lesions on two effects of context after fear extinction

Three conditioned suppression experiments with rats examined the role of the hippocampus in 2 effects of context after extinction. Reinstatement is the context-specific recovery of fear to an extinguished conditioned stimulus (CS) that occurs following independent presentations of the unconditioned stimulus (US), after extinction. Renewal is the recovery of fear when the CS is presented in the context in which it was conditioned, after extinction in a different context. Results indicated that neurotoxic lesions of the hippocampus, performed before conditioning, abolished reinstatement, which depends on context-US associations, but not renewal, which does not. This dissociation is not the result of differences in the recentness of context learning that ordinarily governs the 2 effects. The results suggest that the hippocampus is necessary for some, but not all, types of contextual learning.     

Cardiac defense response as a predictor of fear learning

Prior studies have demonstrated that differences in activation of the defensive motivational system – as indexed by cardiac responses to the CS+ during aversive conditioning – are related to differences in the acquisition of two-levels-of-learning: cognitive (contingency learning) and emotional (fear learning). Here we further explored these differences using an independent psychophysiological test to assess cardiac reactivity – Cardiac Defense Response (CDR) – prior to the aversive conditioning task. Participants were then classified as accelerators or decelerators based on the CDR second accelerative component. Both groups showed contingency learning, as indexed by greater skin conductance changes to CS+ than to CS− during acquisition and by consistent contingency awareness ratings after the conditioning task. However, only accelerators showed affective fear learning, as indexed by greater blinks to CS+ than to CS− during (acquisition) and after (extinction) aversive conditioning. These results extend evidence about differences in the two-levels-of-learning in aversive conditioning as a function of defensive reactivity, and suggest that the CDR second accelerative component could be a reliable predictor of fear learning.