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Despite the great relevance of the neurosteroid allopregnanolone and related isomers to various health conditions, quantification typically involves immunoassay, which suffers from serious issues with cross-reactivity of closely related molecules. This article describes the development and partial validation of a liquid chromatography coupled with tandem mass spectrometry assay for the simultaneous quantification of allopregnanolone, pregnanolone, isopregnanolone, epi-allopregnanolone, and testosterone in the human serum of healthy males and females aged 5-85 years. 1-amino-4-methylpiperazine (AMP) was used as a derivatisation reagent to enhance the ionisation signal. Linearity was calculated at 0.99 with a lower limit of quantification of 10.08 pg/mL for allopregnanolone, along with a linearity of 0.98 and a lower limit of quantification of 42.32 pg/mL for testosterone. Application of the method showed sex and age effects across the lifespan for both allopregnanolone and testosterone, whereas a comparative immunoassay for allopregnanolone was not able to detect differences in the same samples. Our partial validation of this method should provide a new tool for researchers to discover the role of allopregnanolone and its isomers in human health, and how it compares to testosterone and sex hormones., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Significant amounts of research have been devoted to treatment of post-traumatic stress disorder (PTSD) and the understanding of its fear and stress-related symptoms. However, current interventions are only effective in 60 % of the patient population. Allopregnanolone has become a topic of interest for PTSD due to its influences on inhibitory neurotransmission and the physiological stress response. This review explores available literature that suggests that allopregnanolone has an influence on (a) chronic stress and anxiety-like symptoms, (b) fear conditioning and contextual fear, and (c) intrusive and emotional memories. A relationship between allopregnanolone and PTSD is suggested, postulating that allopregnanolone is a potential target for the treatment of PTSD. This very exciting prospect calls for the expansion of research investigating a direct relationship between allopregnanolone and PTSD., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Inc.)

Meta-analyses indicate differences in Pavlovian fear responses between anxious and non-anxious individuals using electrodermal activity (EDA). Recent research, however, has cast doubt on whether these effects are robust to different analytic choices. Using the multiverse approach conceived by Steegen et al. (2016), we surveyed analytic choices typically implemented in clinical fear conditioning research by conducting 1240 analyses reflecting different choice permutations. Only 1.45% of our analyses produced theoretically congruent statistically significant effects, and the strength and direction of the estimated effects varied substantially across EDA processing methods. We conclude that EDA-estimated fear learning differences are vulnerable to researcher degrees of freedom and make recommendations regarding which analytical choices should be approached with a high degree of caution., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Presenting unpaired unconditional stimuli (US) during extinction training reduces the renewal of conditional fear due to context change and slows re-acquisition. The present study investigated whether this reduced return of fear is mediated by Pavlovian inhibitory conditioning to the conditional stimulus paired with the US during acquisition (CS+) that is acquired when this stimulus is presented without the US in an excitatory extinction context. Using an ABA renewal paradigm that trained extinction in a context different from acquisition and renewal test, participants either received no USs (Standard), five unsignalled US presentations (Unsignalled) or five presentations of the US preceded by a novel, third CS (Signalled) during extinction training. Extinction was followed by tests for renewal and re-acquisition. Replicating previous results, renewal of electrodermal conditional responses was observed in group Standard, but not in group Unsignalled. Signalling the additional USs, and thus reducing context conditioning and the potential for inhibitory conditioning, did not reduce their effect in that renewal was absent in group Signalled. These results are inconsistent with an inhibitory conditioning account of the effects of unpaired US presentations during extinction. A trial sequence learning account or an arousal account may explain the effects of unpaired presentations of the US during extinction., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Endocannabinoids are lipid neurotransmitters that play an important part in human health. Recent methods have found that quantification of endocannabinoids in hair and saliva samples is possible using liquid chromatography paired with tandem mass spectrometry (LC-MS/MS). This chapter describes two simple sample preparation methods that can be used to prepare hair and saliva samples for analysis using LC-MS/MS. Our LC-MS/MS method can be applied to both hair and saliva samples and is sufficiently sensitive for endocannabinoid, as well as steroid hormone, quantification in both of these sample matrices. This chapter provides a comprehensive description of how this can be achieved and provides tips and tricks for troubleshooting problems users may experience., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Skin conductance is a commonly used physiological measure during psychology experiments, such as during fear conditioning. Methods for scoring skin conductance responses (SCRs) are highly heterogeneous, though most researchers agree that manually inspected scores provide the highest quality data when compared to most available fully automated scoring methods. However, manual scoring is extremely time-consuming. We developed a semi-automated scoring program that reduces the time required to process SCR data at a level of quality akin to manual scoring. In contrast to all previous scoring programs, our program enables scoring of first interval response (FIR), second interval response (SIR), and third interval response (TIR) SCRs. Using interclass correlation coefficients (ICCs), Bland-Altman plots and Pareto analysis, we show here that our method is highly reliable and produces data that are almost identical to data that are manually scored and scored using LEDALAB. This software is very easy to use and is freely available to download and modify. We expect that this software will be helpful in reducing the time required to produce high quality FIR, SIR, and TIR skin conductance data for psychology researchers around the world., (© 2024 The Author(s). Psychophysiology published by Wiley Periodicals LLC on behalf of Society for Psychophysiological Research.)

Extinction, the repeated presentation of a conditional stimulus (CS) without the unconditional stimulus (US), is the standard paradigm to reduce conditional responding acquired by the repeated pairing of CS and US in acquisition. However, this reduction of conditional responding is prone to relapse. In rodent fear-conditioning, gradual extinction, the fading out of CS-US pairings during extinction, has been shown to reduce the return of fear. The current study replicated the gradual extinction procedure in human fear conditioning and assessed whether it reduced the return of fear due to ABA renewal and reacquisition. During extinction, one group received standard extinction, a second received gradual extinction (increasing the spacing of USs presented after the 1st, 3rd, 6th, 10th, and 15th CS+ trials), and a third received reversed extinction training (decreasing the spacing of USs presented after the 1st, 6th, 10th, 13th, and 15th CS+ trials). Larger renewal and faster reacquisition of differential electrodermal responses to CS+ and CS- were expected after standard and reversed extinction than after gradual extinction training. The results were inconclusive due to the failure to find extinction of differential electrodermal responses and US expectancy ratings in both gradual and reversed extinction groups. Despite successful extinction in group standard, renewal was only observed in US expectancy. Visualization of US expectancy ratings during extinction suggested that potential identification of the US presentation patterns during extinction in the gradual and reversed groups delayed extinction learning., (© 2024 The Author(s). Psychophysiology published by Wiley Periodicals LLC on behalf of Society for Psychophysiological Research.)

Linoleic acid (LA) is required for neuronal development. We have previously demonstrated sex-specific changes in cardiovascular and hepatic function in rat offspring from mothers consuming a high-LA diet, with some effects associated with reduced LA concentration in the postnatal diet. At this time, the impact of a high-maternal-LA diet on offspring brain development and the potential for the postnatal diet to alter any adverse changes are unknown. Rat offspring from mothers fed low- (LLA) or high-LA (HLA) diets during pregnancy and lactation were weaned at postnatal day 25 (PN25) and fed LLA or HLA diets until sacrifice in adulthood (PN180). In the offspring's brains, the postnatal HLA diet increased docosapentaenoate in males. The maternal HLA diet increased LA, arachidonate, docosapentaenoate, C18:0 dimethylacetal (DMA), C16:0 DMA, C16:0 DMA/C16:0, and C18:0 DMA/C18:0, but decreased eoicosenoate, nervoniate, lignocerate, and oleate in males. Maternal and postnatal HLA diets reduced oleate and vaccenate and had an interaction effect on myristate, palmitoleate, and eicosapentaenoate in males. In females, maternal HLA diet increased eicosadienoate. Postnatal HLA diet increased stearate and docosapentaenoate. Maternal and postnatal HLA diets had an interaction effect on oleate, arachidate, and docosahexaenoic acid (DHA)/omega (n)-6 docosapentaenoic acid (DPA) in females. Postnatal HLA diet decreased DHA/n-6 DPA in males and females. Postnatal HLA diet increased plasma endocannabinoids (arachidonoyl ethanolamide and 2-arachidonoyl glycerol), as well as other N-acyl ethanolamides and testosterone. HLA diet alters brain fatty acids, plasma endocannabinoids, and plasmalogen concentrations in a development-specific and sex-specific manner.

Liquid chromatography paired with tandem mass spectrometry (LC-MS/MS) is the gold standard in measurement of endocannabinoid concentrations in biomatrices. We conducted a systematic review of literature to identify advances in targeted LC-MS/MS methods in the period 2017-2024. We found that LC-MS/MS methods for endocannabinoid quantification are relatively consistent both across time and across biomatrices. Recent advances have primarily been in three areas: (1) sample preparation techniques, specific to the chosen biomatrix; (2) the range of biomatrices tested, recently favoring blood matrices; and (3) the breadth of endocannabinoid and endocannabinoid-like analytes incorporated into assays. This review provides a summary of the recent literature and a guide for researchers looking to establish the best methods for quantifying endocannabinoids in a range of biomatrices., (© 2024 The Author(s). Mass Spectrometry Reviews published by John Wiley & Sons Ltd.)

The endogenous cannabinoid (ECB) system is a small molecule lipid signalling system that is involved in stress response activation and is associated with PTSD, but it is unclear whether salivary ECBs are part of the sympathetic nervous system response to stress. We conducted an adapted trauma film paradigm, where participants completed a cold pressor test (or control) while watching a 10-minute trauma film. We also collected saliva and hair samples and tested them for ECBs, cortisol, and salivary alpha amylase (sAA). As hypothesised, there were significant positive correlations between sAA activity and salivary ECB levels, particularly 2-arachidonoyl glycerol (2-AG), though ECBs were not correlated with sAA stress reactivity. Participants who had a significant cortisol response to the trauma film/stressor reported less intrusive memories, which were also less distressing and less vivid. This effect was moderated by arachidonoyl ethanolamide (AEA), where decreases in AEA post-stress were associated with more intrusive memories in cortisol non-responders only. This study provides new evidence for the role of ECBs in the sympathetic nervous system., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The emotional conflict task measures emotional conflict resolution and adaptation, but some studies are unable to find resolution or adaptation effects using this task. We examined boundary conditions and replicability of the emotional conflict resolution and adaptation effects through secondary data analysis, systematic review, and meta-analysis of studies in the field. In our data, we were unable to fully replicate the emotional conflict resolution or adaptation effects and found that most studies using this task ( n = 94) do not report analysis of emotional conflict resolution, with only 28% ( n = 26) studies doing so. Our meta-analysis suggests that studies reporting emotional conflict resolution and adaptation analyses overall report significant but small effects, suggesting the effect is difficult to consistently replicate. Our meta-analysis revealed that controlling for contingency learning may impact the ability of studies to identify conflict resolution. These findings have implications for assessment and interpretation of the emotional conflict task. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Renewal is a 'return of fear' manipulation in human fear conditioning to investigate learning processes underlying anxiety and trauma. Even though renewal paradigms are widely used, no study has compared the strength of different renewal paradigms. We conduct a systematic review (N = 80) and meta-analysis (N = 23) of human fear conditioning studies assessing renewal. Our analysis shows that the classic ABA design is the most effective paradigm, compared to ABC and ABBA designs. We present evidence that conducting extinction in multiple contexts and increasing the similarity between acquisition and extinction contexts reduce renewal. Furthermore, we show that additional cues can be used as safety and 'protection from extinction' cues. The review shows that alcohol weakens the extinction process and that older adults appear less sensitive to context changes and thus show less renewal. The large variability in approaches to study renewal in humans suggests that standardisation of fear conditioning procedures across laboratories would be of great benefit to the field., Competing Interests: Conflict of interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The endocannabinoid (ECB) system has recently been considered a potential treatment target for various clinical disorders. However, research around age- and sex-related changes within the ECB system is relatively limited. To improve our understanding of these changes, the current study measured arachidonoyl ethanolamide (AEA), 2-arachidonoyl glycerol (2-AG), oleoylethanolamine (OEA), palmitoylethanolamine (PEA), arachidonic acid (AA), cortisol, and progesterone in pooled serum samples stratified by sex (male and female) and age groups (5-15; 15-30; 30-45; 45-60; 60-75; 85+), using liquid-chromatography tandem mass spectrometry. Serum progesterone levels significantly increased in females of the 15-30 and 30-45 age groups, before declining. Significantly higher cortisol, AEA, 2-AG, OEA, and PEA were found in males and in older age, while significantly higher AA was found in females. Our results indicate that ECBs and related hormones exhibit sexual dimorphism in the age ranges that correspond with female pregnancy, menopause, and post menopause. Male testosterone levels most likely influences male ECB changes throughout the lifespan. Future research could capitalise on these findings by performing repeated measurements in individuals in a longitudinal style, to further refine the temporal profile of age-specific changes to the ECB system identified here., (© 2024. The Author(s).)

Fear conditioning is a significant area of research that has featured prominently among the topics published in Biological Psychology over the last 50 years. This work has greatly contributed to our understanding of human anxiety and stressor-related disorders. While mainly conducted in the laboratory, recently, there have been initial attempts to conduct fear conditioning experiments online, with around 10 studies published on the subject, primarily in the last two years. These studies have demonstrated the potential of online fear conditioning research, although challenges to ensure that this research meets the same methodological standards as in-person experimentation remain, despite recent progress. We expect that in the coming years new outcome measures will become available online including the measurement of eye-tracking, pupillometry and probe reaction time and that compliance monitoring will be improved. This exciting new approach opens new possibilities for large-scale data collection among hard-to-reach populations and has the potential to transform the future of fear conditioning research., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent studies have shown that fear conditioning experiments can be successfully conducted online. However, there is limited evidence that measures other than subjective ratings of threat expectancy can be collected, which means that online research may not be able to adequately replace laboratory experiments. In the current study, we conducted an online fear conditioning experiment consisting of habituation, acquisition, extinction and 48 h delayed extinction recall using ratings of threat expectancy and conditional stimulus pleasantness, and probe reaction time as outcome measures. The conditional stimuli were categories of words and a levels of processing manipulation explored whether words that were processed at a deeper level during extinction evoked smaller differential threat responses during extinction recall. Although the levels of processing manipulation did not produce a significant outcome, we found that extinction recall was successfully operationalised in our study. Reaction time indicated differential responding during both acquisition and extinction recall, and age of participants was correlated in one of two experiments with differential threat expectancy and reaction time, such that older participants showed better safety learning. The outcomes of this experiment provide multiple novel tools for researchers to explore fear conditioning, especially in an online environment., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

On the basis of substantial preclinical evidence, the endogenous cannabinoid system has been proposed to be closely involved in stress reactivity and extinction of fear. Existing human research supports this proposal to some extent, but existing studies have used only a narrow range of tools and biomatrices to measure endocannabinoids during stress and fear experiments. In the present study we collected hair and saliva samples from 99 healthy participants who completed a fear conditioning and intrusive memory task. Subjective, physiological and biological stress reactivity to a trauma film, which later served as unconditional stimulus during fear conditioning, was also measured. We found that salivary endocannabinoid concentrations predicted subjective responses to stress, but not cortisol stress reactivity, and replicated previous findings demonstrating a sex dimorphism in hair and salivary endocannabinoid levels. Hair 2-arachidonoyl glycerol levels were significantly associated with better retention of safety learning during extinction and renewal phases of fear conditioning, while hair concentrations of oleoylethanolamide and palmitoylethanolamide were associated with overall physiological arousal, but not conditional learning, during fear conditioning. This study is the first to test the relationship between hair and salivary endocannabinoids and these important psychological processes. Our results suggest that these measures may serve as biomarkers of dysregulation in human fear memory and stress., Competing Interests: Declaration of Competing Interest Luke Ney has previously received research funding from E3D Pharma a division of CannaPacific Pty Ltd. He no longer consults for this company and this study was not funded, communicated about, or influenced by E3D Pharma in any way., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The replicability of fear conditioning research has come under recent scrutiny, with increasing acknowledgment that the use of differing materials and methods may lead to incongruent results. Direct comparisons between the main two unconditional stimuli used in fear conditioning - an electric shock or a loud scream-are scarce, and yet these stimuli are usually used interchangeably. In the present study, we tested whether a scream, a shock, or an unpredictable combination of the two affected fear acquisition, extinction, and return of fear amongst healthy participants (N = 109, 81 female). We also collected hair samples and tested the relationship between fear conditioning and hair endocannabinoid levels. Our findings suggest that, although subjective ratings of pleasantness, arousal, and anxiety were similar regardless of the unconditional stimuli used, skin conductance responses were significantly lower for stimuli paired with the scream compared to a shock alone. Further, reducing the predictability of the unconditional stimulus reduced habituation of skin conductance responses during acquisition and reacquisition, but did not produce stronger conditioning compared to shock alone. Exploratory analyses suggested that hair endocannabinoids were associated with overall physiological arousal during fear conditioning, as well as higher return of fear to the threat cue, but not to the safety cue. These findings have multiple implications for the design and replicability of fear conditioning research and provide the first evidence for an association between hair levels of endocannabinoids and human fear conditioning., (© 2023 The Authors. Psychophysiology published by Wiley Periodicals LLC on behalf of Society for Psychophysiological Research.)

Introduction: Preclinical and experimental research have provided promising evidence that medicinal cannabis may be efficacious in the treatment of posttraumatic stress disorder (PTSD). However, implementation of medicinal cannabis into routine clinical therapies may not be straightforward., Areas Covered: In this review, we describe some of the clinical, practical, and safety challenges that must be addressed for cannabis-based treatment of PTSD to be feasible in a real-world setting. These issues are especially prevalent if medicinal cannabis is to be combined with trauma-focused psychotherapy., Expert Opinion: Future consideration of the clinical and practical considerations of cannabis use in PTSD therapy will be essential to both the efficacy and safety of the treatment protocols that are being developed. These issues include dose timing and titration, potential for addiction, product formulation, windows of intervention, and route of administration. In particular, exposure therapy for PTSD involves recall of intense emotions, and the interaction between cannabis use and reliving of trauma memories must be explored in terms of patient safety and impact on therapeutic outcomes.

Dysregulated consolidation of emotional memories is a core feature of posttraumatic stress disorder (PTSD). Brain Derived Neurotrophic Factor (BDNF) influences synaptic plasticity and emotional memory consolidation. The BDNF Val66Met polymorphism has been associated with PTSD risk and memory deficits respectively, although findings have been inconsistent, potentially due to a failure to control for important confounds such as sex, ethnicity, and the timing/extent of previous trauma experiences. Furthermore, very little research has examined the impact of BDNF genotypes on emotional memory in PTSD populations. This study investigated the interaction effects of Val66Met and PTSD symptomatology in an emotional recognition memory task in 234 participants divided into healthy control (n = 85), trauma exposed (TE: n = 105) and PTSD (n = 44) groups. Key findings revealed impaired negative recognition memory in PTSD compared to control and TE groups and in participants with the Val/Met compared to the Val/Val genotype. There was a group × genotype interaction showing no Met effect in the TE group despite significant effects in PTSD and controls. Results suggest that people previously exposed to trauma who do not develop PTSD may be protected from the BDNF Met effect, however more research is needed to replicate findings and to explore the epigenetic and neural processes involved., (© 2023. The Author(s).)

Sleep has been found to play a key role in fear conditioning, extinction learning and extinction recall, and sleep disturbances are linked to many mental disorders including post-traumatic stress disorder (PTSD). Previous studies examining associations between sleep and fear or extinction processes primarily focused on objectively measured sleep architecture. Little research has so far focused on subjective sleep measures and particularly in clinical populations, which often experience subjectively poor sleep, including PTSD. Here we investigated whether subjective sleep disturbance, sleep onset latency, wake after sleep onset or sleep efficiency were related to fear conditioning, extinction learning or extinction recall in a large sample of individuals with a range of PTSD symptom severity (n = 248). Overall, we did not find that subjective sleep was associated with fear conditioning or extinction processes. However, exploratory analyses examining the moderating effect of sex found that shorter sleep onset latency and greater sleep efficiency were associated with improved extinction recall in women with higher PTSD symptom severity. This suggests that less time falling asleep and longer time asleep while in bed may be protective in highly symptomatic women against the commonly observed impaired extinction recall in PTSD. More studies are needed to explore sex-specific effects further., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Past fear conditioning studies have used different types of conditional stimuli (CSs). Whether this choice affects learning outcomes in particular when neutral stimuli (e.g., neutral faces vs. shapes) are used is unclear. Data were aggregated across nine studies using an electric shock unconditional stimulus to test for differences in acquisition and extinction of electrodermal responses and self-reported CS pleasantness when CSs were neutral faces or shapes (Experiment 1, N = 594) and when CSs were angry or neutral faces (Experiment 2, N = 157). Reliable electrodermal conditioning was observed in all stimulus conditions. We found stronger differential conditioning in electrodermal second interval responses and CS pleasantness and more pronounced extinction in CS pleasantness for neutral shape than neutral face CSs, but no differences in electrodermal first interval responses, the most frequently reported index of fear conditioning. For angry and neutral face CSs, there were no differences during acquisition, but the extinction of first and second interval electrodermal conditioning to angry faces was retarded relative to neutral faces. Acquisition of differential CS pleasantness, which was reliably observed for neutral face CSs, was absent for angry face CSs. The current results suggest that fear conditioning with a neutral face and shape CSs yields broadly similar results with differences limited to second interval electrodermal responses and CS pleasantness ratings. Using angry face CSs resulted in impaired extinction of electrodermal indices and no differential CS pleasantness ratings and should only be considered in studies designed to address questions about these specific CS materials., (© 2022 The Authors. Psychophysiology published by Wiley Periodicals LLC on behalf of Society for Psychophysiological Research.)

Past research has shown that Intolerance of Uncertainty (IU) affects Pavlovian fear conditioning processes. In particular, extinction of learned fear is delayed in those reporting high IU. Reports of differences during acquisition are less consistent with most of the studies reporting no evidence for effects of IU. This may be due to past studies' focus on first interval electrodermal responses or fear potentiated startle, rather than on indices that may better capture uncertainty - like the response to the absence of a probabilistic unconditional stimulus. The current analysis combined data across three experiments that employed a 50 % reinforcement schedule and assessed electrodermal responses and (in two experiments) ratings of conditional stimulus pleasantness. Participants scoring high on IU showed overall larger electrodermal first interval responses during habituation and acquisition but did not differ from those scoring low on IU in differential conditioning (the difference between CS+ and CS-), as indicated by electrodermal first or second interval responses or ratings of CS pleasantness. However, participants high in IU showed larger differential third interval electrodermal responses to the omission of the electro-tactile unconditional stimulus during acquisition. Some evidence for this difference emerged in each experiment, supporting the reliability of the result. The current results suggest that effects of IU emerge in conditions of high uncertainty in Pavlovian fear learning tasks, such as during the omission of probabilistic unconditional stimuli., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Some previous research has shown stronger acquisition and impaired extinction of fear conditioned to angry or fearful compared to happy or neutral face conditional stimuli (CS) - a difference attributed to biological 'preparedness'. A systematic review and meta-analysis of fear conditioning studies comparing face CSs of differing expressions identified thirty studies, eighteen of which were eligible for meta-analysis. Skin conductance responses were larger to angry or fearful faces compared to happy or neutral faces during habituation, acquisition and extinction. Significant differences in differential conditioning between angry, fearful, neutral, and happy face CSs were also found, but differences were more prominent between angry and neutral faces compared to angry/fearful and happy faces. This is likely due to lower arousal elicited by neutral compared to happy faces, which may be more salient as CSs. The findings suggest there are small to moderate differences in differential conditioning when angry or fearful compared to happy or neutral faces are used as CSs. These findings have implications for fear conditioning study design and the preparedness theory., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Fear conditioning paradigms are critical to understanding anxiety-related disorders, but studies use an inconsistent array of methods to quantify the same underlying learning process. We previously demonstrated that selection of trials from different stages of experimental phases and inconsistent use of average compared to trial-by-trial analysis can deliver significantly divergent outcomes, regardless of whether the data is analysed with extinction as a single effect, as a learning process over the course of the experiment, or in relation to acquisition learning. Since small sample sizes are attributed as sources of poor replicability in psychological science, in this study we aimed to investigate if changes in sample size influences the divergences that occur when different kinds of fear conditioning analyses are used. We analysed a large data set of fear acquisition and extinction learning (N = 379), measured via skin conductance responses (SCRs), which was resampled with replacement to create a wide range of bootstrapped databases (N = 30, N = 60, N = 120, N = 180, N = 240, N = 360, N = 480, N = 600, N = 720, N = 840, N = 960, N = 1080, N = 1200, N = 1500, N = 1750, N = 2000) and tested whether use of different analyses continued to produce deviating outcomes. We found that sample size did not significantly influence the effects of inconsistent analytic strategy when no group-level effect was included but found strategy-dependent effects when group-level effects were simulated. These findings suggest that confounds incurred by inconsistent analyses remain stable in the face of sample size variation, but only under specific circumstances with overall robustness strongly hinging on the relationship between experimental design and choice of analyses. This supports the view that such variations reflect a more fundamental confound in psychological science-the measurement of a single process by multiple methods., Competing Interests: The authors have declared that no competing interests exist.

A growing literature has sought to combine fear conditioning paradigms with the trauma film paradigm to study the associative learning properties of intrusive re-experiencing in PTSD. We review this innovative approach and the recent findings by highlighting their relevance to cognitive and conditioning theories of PTSD. We also conduct a meta-analysis of the available studies to demonstrate that, for most outcome measures, fear learning using a traumatic film clip unconditional stimulus yields results similar to those seen with an electro-tactile unconditional stimulus, which implies that the combined paradigm shares at least some properties of more standard fear conditioning paradigms. We argue that careful use of this combined paradigm will provide important new insights into the mechanisms underlying memory symptoms of PTSD and will allow rigorous testing of cognitive theories of the disorder., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The endocannabinoid system is known to be involved in mechanisms relevant to PTSD aetiology and maintenance, though this understanding is mostly based on animal models of the disorder. Here we review how human paradigms can successfully translate animal findings to human subjects, with the view that substantially increased insight into the effect of endocannabinoid signalling on stress responding, emotional and intrusive memories, and fear extinction can be gained using modern paradigms and methods for assessing the state of the endocannabinoid system in PTSD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Anxiety and stress-related disorders are more prevalent in women and associated with negative emotional memory consolidation as well as impaired fear extinction recall. Recent research has identified a role of gonadal steroid hormones in influencing emotional memories and fear extinction, however most individual studies have small samples and employed various protocols. A systematic review and meta-analysis were conducted on studies that examined sex hormones (estrogen, progesterone, testosterone, allopregnanolone, dehydroepiandrosterone) on four aspects of memory, namely, intentional recall (k = 13), recognition memory (k = 7), intrusive memories (k = 9), and extinction recall (k = 11). The meta-analysis on natural cycling women revealed that progesterone level was positively associated with negative recall and negative intrusive memories, and this effect on intentional recall was enhanced under stress induction. Estradiol level was positively associated with extinction recall. This study reveals an important role of progesterone and estradiol in influencing emotional memory consolidation. It highlights the need to control for these hormonal effects and examine progesterone and estradiol concurrently across all menstrual phases in future emotional memory paradigms., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The endocannabinoid system is gaining increasing attention as a favorable target for improving posttraumatic stress disorder (PTSD) treatments. Exposure therapy is the gold-standard treatment for PTSD, and fear extinction learning is a key concept underlying successful exposure., Methods: This study examined the role of genetic endocannabinoid polymorphisms in a fear extinction paradigm with PTSD compared to healthy participants (N = 220). Participants provided saliva for genotyping, completed a fear conditioning and extinction task, with blood samples taken before and after the task (n = 57). Skin conductance was the outcome and was analyzed using mixed models., Results: Results for cannabinoid receptor type 1 polymorphisms suggested that minor alleles of rs2180619 and rs1049353 were associated with poorer extinction learning in PTSD participants. The minor allele of the fatty acid amide hydrolase (FAAH) polymorphism rs324420 was associated with worse extinction in PTSD participants. Subanalysis of healthy participants (n = 57) showed the FAAH rs324420 genotype effect was dependent on plasma arachidonoyl ethanolamide (AEA) level, but not oleoylethanolamide or 2-arachidonoyl glycerol. Specifically, higher but not lower AEA levels in conjunction with the minor allele of FAAH rs324420 were associated with better extinction learning., Conclusions: These findings provide translational evidence that cannabinoid receptor 1 and AEA are involved in extinction learning in humans. FAAH rs324420's effect on fear extinction is moderated by AEA plasma level in healthy controls. These findings imply that FAAH inhibitors may be effective for targeting anxiety in PTSD, but this effect needs to be explored further in clinical populations., (© 2021 Wiley Periodicals LLC.)

Brain-derived neurotropic factor (BDNF) is a potent regulator of memory processes and is believed to influence the consolidation of fear extinction memories. No previous human study has tested the effect of unstimulated BDNF on fear extinction recall, and no study has tested the association between plasma BDNF levels and psychophysiological responding during an extinction paradigm. We tested the association between fear responses during a 2-day differential conditioning, extinction and extinction recall paradigm and Val66Met genotype in a group of healthy participants (N = 191). There were no group differences during habituation or acquisition. Met allele carriers compared to Val homozygotes displayed higher responses to the CS + compared to the CS- during extinction learning and had higher responding to both the CS+ and CS- during extinction recall. Plasma levels of BDNF protein that were collected in a sub-sample of the group (n = 56) moderated the effect of Met allele presence, such that lower BDNF level was associated with higher skin conductance response in the Met but not Val group to the CS+ during extinction learning and to both the CS+ and CS- during extinction recall. The current results extend previous observations of a Val66Met effect during fear extinction learning to extinction recall and show for the first time that these effects are moderated by plasma BDNF level., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Sleep may contribute to the long-lasting consolidation and processing of emotional memories. Experimental fear conditioning and extinction paradigms model the development, maintenance, and treatment of anxiety disorders. The literature provides compelling evidence for the involvement of rapid eye movement (REM) sleep in the consolidation of such memories. This meta-analysis correlated polysomnographic sleep findings with psychophysiological reactivity to the danger (CS+) and safety stimuli (CS-), to clarify the specific role of sleep stages before and after fear conditioning, extinction learning and extinction recall. Overall, there was evidence that more pre-learning sleep stage two and less slow wave sleep was associated with higher psychophysiological reactivity to the safety stimulus during extinction learning. Preliminary evidence found here support the role of REM sleep during the post-extinction consolidation sleep phase in clinical populations with disrupted sleep, but not in healthy controls. Furthermore, the meta-regressions found that sex moderated the associations between sleep and psychophysiological reactivity throughout the paradigm providing evidence for diverging correlations in male and females. Specifically, increased post-extinction REM was associated with poorer extinction and safety recall in females while the opposite was found in males. These results have implications for future research in the role of sleep in emotional memory processing., Competing Interests: Conflicts of interest Maya Schenker was funded by the Melbourne Graduate Research Scholarship. The authors have no other conflict of interest to disclose., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Understanding the role of endogenous cannabinoids (endocannabinoids) in disease is of increasing importance. However, tools to investigate endocannabinoid levels in humans are limited. In the current study, we report a simplified sample preparation method for quantifying endocannabinoids and steroid hormones in hair using liquid-liquid extraction combined with ultra performance liquid chromatography coupled to tandem mass spectrometry. The fully validated method is at least R 2  = 0.99 linear between 5 and 1,000 pg/mg for each analyte and the detection limits are at or below 0.50 pg/mg for cortisol, progesterone, oleoylethanolamide, and arachidonoyl ethanolamide, and 2.65 pg/mg for 2-arachidonoyl glycerol. Sequential extraction of hair samples revealed that multiple extractions may be required for quantitative recovery of steroids. However endogenous cannabinoids were efficiently recovered using a single sample extraction. The method was applied to a psychosocial stress study where participants provided samples of both hair and saliva. Endogenous hair arachidonoyl ethanolamide levels were negatively associated with resting, but not stressed, salivary cortisol levels in healthy participants. This simplified method enables the detailed study of hormonal and endocannabinoids in human hair with high sensitivity., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Low levels of estradiol in women have been associated with impaired fear extinction recall, with suggestions this may promote the return of fear and heighten the female vulnerability for anxiety disorders. A particularly important measure for the return of fear is reinstatement, but no human studies to date have examined the impact of estradiol on fear reinstatement. Forty-two healthy females completed a differential fear conditioning, extinction and reinstatement task with skin conductance response (SCR) amplitude indexing level of conditioned fear. Saliva samples were taken to measure estradiol and progesterone. To examine fear reinstatement, SCR amplitude was compared between the last trial of the late extinction phase to the first re-extinction trial following the unsignaled presentation of two aversive electric shocks. No significant effects of estradiol were found for acquisition of fear conditioning or fear extinction learning. Lower estradiol predicted a significantly larger generalized SCR amplitude at re-extinction (post-reinstatement) in women. This provides novel evidence suggesting a protective role of estradiol in potentially reducing the relapse of fear following re-exposure to aversive stimuli, although further research is necessary in clinical populations to clarify this effect., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

There currently exist few frameworks for common neurobiology between reexperiencing and negative cognitions and mood symptoms of PTSD. Adopting a dopaminergic framework for PTSD unites many aspects of unique symptom clusters, and this approach also links PTSD symptomology to common comorbidities with a common neurobiological deficiency. Here we review the dopamine literature and incorporate it with a growing field of research that describes both the contribution of endocannabinoids to fear extinction and PTSD, as well as the interactions between dopaminergic and endocannabinoid systems underlying this disorder. Based on current evidence, we outline an early, preliminary model that links re-experiencing and negative cognitions and mood in PTSD by invoking the interaction between endocannabinoid and dopaminergic signalling in the brain. These interactions between PTSD, dopamine and endocannabinoids may have implications for future therapies for treatment-resistant and comorbid PTSD patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background : PTSD is characterised by severe sleep disturbances, which is increasingly recognised to in many cases consist of similar symptomology to sleep disorders such as REM Behaviour Disorder (RBD). The present study aimed to investigate whether different aspects of sleep quality influence intrusive memory development and whether PTSD status moderates this relationship. Participants and Methods : 34 PTSD, 52 trauma-exposed (TE) and 42 non-trauma exposed (NTE) participants completed an emotional memory task, where they viewed 60 images (20 positive, 20 negative and 20 neutral) and, two days later, reported how many intrusive memories they had of each valence category. Participants also completed three measures of sleep quality: the Pittsburgh Sleep Quality Index, the REM Behaviour Disorder Screening Questionnaire and total hours slept before each session. Results : The PTSD group reported poorer sleep quality than both TE and NTE groups on all three measures, and significantly more negative intrusive memories than the NTE group. Mediation analyses revealed that self-reported RBD symptomology before the second session mediated the relationship between PTSD status and intrusive memories. Follow-up moderation analyses revealed that self-reported RBD symptomology before the second session was only a significant predictor of intrusion in the PTSD group, though with a small effect size. Conclusions : These findings suggest that RBD symptomology is an indicator of consolidation of intrusive memories in PTSD but not trauma-exposed or healthy participants, which supports the relevance of characterising RBD in PTSD.

Cannabis sativa ( Cannabis ) is one of the world's most well-known, yet maligned plant species. However, significant recent research is starting to unveil the potential of Cannabis to produce secondary compounds that may offer a suite of medical benefits, elevating this unique plant species from its illicit narcotic status into a genuine biopharmaceutical. This review summarises the lengthy history of Cannabis and details the molecular pathways that underpin the production of key secondary metabolites that may confer medical efficacy. We also provide an up-to-date summary of the molecular targets and potential of the relatively unknown minor compounds offered by the Cannabis plant. Furthermore, we detail the recent advances in plant science, as well as synthetic biology, and the pharmacology surrounding Cannabis. Given the relative infancy of Cannabis research, we go on to highlight the parallels to previous research conducted in another medically relevant and versatile plant, Papaver somniferum (opium poppy), as an indicator of the possible future direction of Cannabis plant biology. Overall, this review highlights the future directions of cannabis research outside of the medical biology aspects of its well-characterised constituents and explores additional avenues for the potential improvement of the medical potential of the Cannabis plant.

Background: Transcranial direct current stimulation (tDCS) has previously been shown to improve fear extinction learning and retention when administered prior to or during extinction learning. This study investigates whether tDCS immediately following extinction learning improves efficacy of extinction memory retention., Methods: 30 participants completed a 2-day fear learning and extinction paradigm, where they acquired fear of a stimulus conditioned to an aversive electric shock on day 1. Extinction learning occurred on day 1, with tDCS or sham tDCS administered immediately following the learning phase. Participants returned for a second day test of extinction memory recall. Skin conductance was measured as the primary outcome., Results/conclusions: Participants in the tDCS group showed impaired fear extinction retention on day 2, marked by significant generalisation of fear to the safety stimulus. This contrasts with earlier studies showing improved extinction retention when stimulation occurred during encoding of extinction learning, compared to immediate consolidation as in our study. These findings may have important implications for the use of tDCS during exposure therapy for anxiety and trauma disorders., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Immunoassay overestimates progesterone in blood, but no studies have tested whether this occurs in saliva. We measured progesterone in saliva using immunoassay and mass spectrometry. We tested the immunoassay for cross reactivity with dehydroepiandrosterone sulfate (DHEA-S) and 17α-hydroxyprogesterone (17α-OHP). Progesterone was significantly higher in immunoassay compared to mass spectrometry. Immunoassay progesterone levels increased in when incremental levels of 17α-OHP standard was added. This effect was not observed with the addition of DHEA-S. Research using salivary progesterone immunoassay techniques should be wary, particularly with individuals taking steroid supplementation or with high levels of progesterone metabolites., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Robustness of fear conditioning and extinction paradigms has become increasingly important for many researchers interested in improving the study of anxiety and trauma disorders. We recently illustrated the wide variability in data analysis techniques in this paradigm, which we argued may result in a lack of robustness. In the current study, we resampled data from six of our own fear acquisition and extinction data sets, with skin conductance as the outcome. In the resampled and original data sets, we found that effect sizes that were calculated using discrepant statistical strategies, sourced from a non-exhaustive search of high-impact articles, were often poorly correlated. The main contributors to poor correlations were the selection of trials from different stages of each experimental phase and the use of average compared to trial-by-trial analysis. These findings reinforce the importance of focusing on robustness in the psychophysiological measurement of fear acquisition and extinction in the laboratory and may guide prospective researchers in which decisions may most impact the robustness of their results., (© 2020 Society for Psychophysiological Research.)