Acetylcholine and metacognition during sleep.

Acetylcholine is a neurotransmitter and neuromodulator involved in a variety of cognitive functions. Additionally, acetylcholine is involved in the regulation of REM sleep: cholinergic neurons in the brainstem and basal forebrain project to and innervate wide areas of the cerebral cortex, and reciprocally interact with other neuromodulatory systems, to produce the sleep-wake cycle and different sleep stages. Consciousness and cognition vary considerably across and within sleep stages, with metacognitive capacity being strikingly reduced even during aesthetically and emotionally rich dream experiences. A notable exception is the phenomenon of lucid dreaming-a rare state whereby waking levels of metacognitive awareness are restored during sleep-resulting in individuals becoming aware of the fact that they are dreaming. The role of neurotransmitters in these fluctuations of consciousness and cognition during sleep is still poorly understood. While recent studies using acetylcholinesterase inhibitors suggest a potential role of acetylcholine in the occurrence of lucid dreaming, the underlying mechanisms by which this effect is produced remains un-modelled and unknown; with the causal link between cholinergic mechanisms and upstream psychological states being complex and elusive. Several theories and approaches targeting the association between acetylcholine and metacognition during wakefulness and sleep are highlighted in this review, moving through microscopic, mesoscopic and macroscopic levels of analysis to detail this phenomenon at several organisational scales. Several exploratory hypotheses will be developed to guide future research towards fully articulating how metacognition is affected by activity at the acetylcholine receptor.

Observation of others' threat reactions recovers memories previously shaped by firsthand experiences.

Information about dangers can spread effectively by observation of others' threat responses. Yet, it is unclear if such observational threat information interacts with associative memories that are shaped by the individual's direct, firsthand experiences. Here, we show in humans and rats that the mere observation of a conspecific's threat reactions reinstates previously learned and extinguished threat responses in the observer. In two experiments, human participants displayed elevated physiological responses to threat-conditioned cues after observational reinstatement in a context-specific manner. The elevation of physiological responses (arousal) was further specific to the context that was observed as dangerous. An analogous experiment in rats provided converging results by demonstrating reinstatement of defensive behavior after observing another rat's threat reactions. Taken together, our findings provide cross-species evidence that observation of others' threat reactions can recover associations previously shaped by direct, firsthand aversive experiences. Our study offers a perspective on how retrieval of threat memories draws from associative mechanisms that might underlie both observations of others' and firsthand experiences.

Effective or predatory funding? Evaluating the hidden costs of grant applications.

Researchers are spending an increasing fraction of their time on applying for funding; however, the current funding system has considerable deficiencies in reliably evaluating the merit of research proposals, despite extensive efforts on the sides of applicants, grant reviewers and decision committees. For some funding schemes, the systemic costs of the application process as a whole can even outweigh the granted resources-a phenomenon that could be considered as predatory funding. We present five recommendations to remedy this unsatisfactory situation.

Effect of Intraoperative Handovers of Anesthesia Care on Mortality, Readmission, or Postoperative Complications Among Adults: The HandiCAP Randomized Clinical Trial.

Importance: Intraoperative handovers of anesthesia care are common. Handovers might improve care by reducing physician fatigue, but there is also an inherent risk of losing critical information. Large observational analyses report associations between handover of anesthesia care and adverse events, including higher mortality. Objective: To determine the effect of handovers of anesthesia care on postoperative morbidity and mortality. Design, Setting, and Participants: This was a parallel-group, randomized clinical trial conducted in 12 German centers with patients enrolled between June 2019 and June 2021 (final follow-up, July 31, 2021). Eligible participants had an American Society of Anesthesiologists physical status 3 or 4 and were scheduled for major inpatient surgery expected to last at least 2 hours. Interventions: A total of 1817 participants were randomized to receive either a complete handover to receive anesthesia care by another clinician (n = 908) or no handover of anesthesia care (n = 909). None of the participating institutions used a standardized handover protocol. Main Outcomes and Measures: The primary outcome was a 30-day composite of all-cause mortality, hospital readmission, or serious postoperative complications. There were 19 secondary outcomes, including the components of the primary composite, along with intensive care unit and hospital lengths of stay. Results: Among 1817 randomized patients, 1772 (98%; mean age, 66 [SD, 12] years; 997 men [56%]; and 1717 [97%] with an American Society of Anesthesiologists physical status of 3) completed the trial. The median total duration of anesthesia was 267 minutes (IQR, 206-351 minutes), and the median time from start of anesthesia to first handover was 144 minutes in the handover group (IQR, 105-213 minutes). The composite primary outcome occurred in 268 of 891 patients (30%) in the handover group and in 284 of 881 (33%) in the no handover group (absolute risk difference [RD], -2.5%; 95% CI, -6.8% to 1.9%; odds ratio [OR], 0.89; 95% CI, 0.72 to 1.10; P = .27). Nineteen of 889 patients (2.1%) in the handover group and 30 of 873 (3.4%) in the no handover group experienced all-cause 30-day mortality (absolute RD, -1.3%; 95% CI, -2.8% to 0.2%; OR, 0.61; 95% CI, 0.34 to 1.10; P = .11); 115 of 888 (13%) vs 136 of 872 (16%) were readmitted to the hospital (absolute RD, -2.7%; 95% CI, -5.9% to 0.6%; OR, 0.80; 95% CI, 0.61 to 1.05; P = .12); and 195 of 890 (22%) vs 189 of 874 (22%) experienced serious postoperative complications (absolute RD, 0.3%; 95% CI, -3.6% to 4.1%; odds ratio, 1.02; 95% CI, 0.81 to 1.28; P = .91). None of the 19 prespecified secondary end points differed significantly. Conclusions and Relevance: Among adults undergoing extended surgical procedures, there was no significant difference between the patients randomized to receive handover of anesthesia care from one clinician to another, compared with the no handover group, in the composite primary outcome of mortality, readmission, or serious postoperative complications within 30 days. Trial Registration: ClinicalTrials.gov Identifier: NCT04016454.

The migraineur's brain networks: Continuous resting state fMRI over 30 days.

OBJECTIVE: The aim of the current study was to identify typical alterations in resting state connectivity within different stages of the migraine cycle and to thus explore task-free mechanisms of headache attack generation in migraineurs. BACKGROUND: Recent evidence in migraine pathophysiology suggests that hours and even days before headache certain changes in brain activity take place, ultimately leading to an attack. Here, we investigate changes before headache onset using resting state functional magnetic resonance imaging (fMRI). METHODS: Nine episodic migraineurs underwent daily resting state functional magnetic resonance imaging for a minimum period of 30 consecutive days, leading to a cumulative number of 282 total days scanned. Thus, data from 15 spontaneous headache attacks were acquired. This allows analysing not only the ictal and the interictal phase of migraine but also the preictal phase. ROI-to-ROI (region of interest) and ROI-to-voxel connectivity was calculated over the migraine cycle. RESULTS: Within the ROI-to-ROI analysis, the right nucleus accumbens showed enhanced functional connectivity to the left amygdala, hippocampus and gyrus parahippocampalis in the preictal phase compared to the interictal phase. ROI-to-voxel connectivity of the right accumbens with the dorsal rostral pons was enhanced during the preictal phase compared to interictally. Regarding custom defined ROIs, the dorsal pons was ictally functionally more strongly coupled to the hypothalamic area than interictally. CONCLUSIONS: This unique data set suggests that particularly connectivity changes in dopaminergic centres and between the dorsal pons and the hypothalamus are important within migraine attack generation and sustainment.

Intact Classical Fear Conditioning to Interpersonally Threatening Stimuli in Borderline Personality Disorder.

Threat hypersensitivity is regarded as a central mechanism of deficient emotion regulation, a core feature of patients with borderline personality disorder (BPD). Here, we employed a classical fear-conditioning protocol in which interpersonally threatening, interpersonally non-threatening, and non-social (neutral) visual stimuli were predictive of an aversive auditory stimulus in a sample of 23 medication-free adult female patients with BPD and 21 age- and IQ-matched healthy women. The results did not confirm the hypothesized enhanced and prolonged conditioned skin conductance responses (SCR) and subjective stress and expectancy ratings to interpersonally threatening stimuli in patients with BPD compared to healthy women. Patients with BPD generally expected the aversive stimulus more often irrespective of stimulus category and conditioning. Furthermore, patients with BPD showed larger conditioned SCR to interpersonally non-threatening and neutral than interpersonally threatening stimuli, while interpersonally threatening stimuli elicited higher SCR compared to non-threatening or neutral stimuli in healthy controls. Together with previous studies, the results suggest no alterations in fear conditioning to generally aversive stimuli in BPD. Further studies using stimuli with BPD-specific topics, such as abandonment or rejection, and/or to investigate more interpersonal forms of learning, such as observational or instructed conditioning, are urgently needed to further elucidate the mechanisms involved in the etiology and maintenance of threat hypersensitivity in BPD.

A common neural network differentially mediates direct and social fear learning.

Across species, fears often spread between individuals through social learning. Yet, little is known about the neural and computational mechanisms underlying social learning. Addressing this question, we compared social and direct (Pavlovian) fear learning showing that they showed indistinguishable behavioral effects, and involved the same cross-modal (self/other) aversive learning network, centered on the amygdala, the anterior insula (AI), and the anterior cingulate cortex (ACC). Crucially, the information flow within this network differed between social and direct fear learning. Dynamic causal modeling combined with reinforcement learning modeling revealed that the amygdala and AI provided input to this network during direct and social learning, respectively. Furthermore, the AI gated learning signals based on surprise (associability), which were conveyed to the ACC, in both learning modalities. Our findings provide insights into the mechanisms underlying social fear learning, with implications for understanding common psychological dysfunctions, such as phobias and other anxiety disorders.

Atypical meningioma: progression-free survival in 161 cases treated at our institution with surgery versus surgery and radiotherapy.

Although atypical meningioma recurs frequently in spite of total resection and/or radiotherapy, no consensus on optimal adjuvant management was found. However, several retrospective studies analysed the additional effect of adjuvant radiotherapy in atypical meningioma with inconsistent results. Therefrom, the purpose of this study was to evaluate prognostic factors influencing the recurrence/progression and progression-free survival (PFS) rates of atypical meningioma, particularly focused on the role of postoperative adjuvant radiotherapy. Between February 2001 and March 2015, 161 atypical meningioma resections were performed in our Department of Neurosurgery, of which, 128 cases underwent surgical treatment alone and 33 cases underwent surgery and radiotherapy. Kaplan-Meier analysis was used to provide median point estimates and PFS rates. The Cox-regression model was used in the univariate and multivariate analysis to identify significant factors associated with treatment. The extent of resection (Simpson grade I and II) significantly influenced the risk of recurrence (hazard ratio = 1.8, CI (95%) 1.3-2.6, p-value = 0.0004). There was no significant benefit for progression-free survival after adjuvant radiotherapy (hazard ratio = 1.48, CI (95%) 0.76-2.86, p-value = 0.22). Additionally, meningioma located at the anterior and posterior fossa showed a significantly longer PFS compared to other locations (p-value = 0.03). Adjuvant postoperative radiotherapy had no significant impact on recurrence/progression rate or PFS. The extent of resection according to Simpson grade remains the most important prognostic factor associated with lower recurrence/progression rates and longer PFS in patients with atypical meningioma. The location of the tumours at the anterior or posterior fossa was an independent factor associated with improved PFS.

An elevated plus-maze in mixed reality for studying human anxiety-related behavior.

BACKGROUND: A dearth of laboratory tests to study actual human approach-avoidance behavior has complicated translational research on anxiety. The elevated plus-maze (EPM) is the gold standard to assess approach-avoidance behavior in rodents. METHODS: Here, we translated the EPM to humans using mixed reality through a combination of virtual and real-world elements. In two validation studies, we observed participants' anxiety on a behavioral, physiological, and subjective level. RESULTS: Participants reported higher anxiety on open arms, avoided open arms, and showed an activation of endogenous stress systems. Participants' with high anxiety exhibited higher avoidance. Moreover, open arm avoidance was moderately predicted by participants' acrophobia and sensation seeking, with opposing influences. In a randomized, double blind, placebo controlled experiment, GABAergic stimulation decreased avoidance of open arms while alpha-2-adrenergic antagonism increased avoidance. CONCLUSION: These findings demonstrate cross-species validity of open arm avoidance as a translational measure of anxiety. We thus introduce the first ecologically valid assay to track actual human approach-avoidance behavior under laboratory conditions.

Dopaminergic receptor blockade changes a functional connectivity network centred on the amygdala.

Resting-state connectivity has become an increasingly important measure in characterizing the functional integrity of brain circuits in neuro-psychiatric conditions. One approach that has recently gained prominence in this regard-and which we use in this study-is to investigate how resting-state connectivity depends on the integrity of certain neuromodulator systems. Here, we use a pharmacological challenge in combination with functional magnetic resonance imaging to investigate the impact of dopaminergic receptor blockade on whole brain functional connectivity in twenty healthy human subjects. Administration of the D2-receptor antagonist haloperidol led to a profound change in functional integration in network nodes linked to the amygdala. Compared to placebo and baseline measurements, network-based statistics and pairwise connectivity analyses revealed reduced connectivity and decreased link strength between the amygdala and the bilateral posterior cingulate cortex and other cortical areas. This was complemented by less extensive but very circumscribed enhanced connectivity between the amygdala and the right putamen during D2-receptor blockade. It will be interesting to investigate whether these pharmacologically induced shifts in resting-state connectivity will similarly be evident in clinical conditions that involve a dysfunction of the dopaminergic system. Our findings might also aid in interpreting alterations in more complex states, such as those seen psychiatric conditions and their treatment. Hum Brain Mapp 37:4148-4157, 2016. © 2016 Wiley Periodicals, Inc.

Single dose of L-dopa makes extinction memories context-independent and prevents the return of fear.

Traumatic events can engender persistent excessive fear responses to trauma reminders that may return even after successful treatment. Extinction, the laboratory analog of behavior therapy, does not erase conditioned fear memories but generates competing, fear-inhibitory "extinction memories" that, however, are tied to the context in which extinction occurred. Accordingly, a dominance of fear over extinction memory expression--and, thus, return of fear--is often observed if extinguished fear stimuli are encountered outside the extinction (therapy) context. We show that postextinction administration of the dopamine precursor L-dopa makes extinction memories context-independent, thus strongly reducing the return of fear in both mice and humans. Reduced fear is accompanied by decreased amygdala and enhanced ventromedial prefrontal cortex activation in both species. In humans, ventromedial prefrontal cortex activity is predicted by enhanced resting-state functional coupling of the area with the dopaminergic midbrain during the postextinction consolidation phase. Our data suggest that dopamine-dependent boosting of extinction memory consolidation is a promising avenue to improving anxiety therapy.

Neural correlates of biased social fear learning and interaction in an intergroup context.

Associations linking a fearful experience to a member of a social group other than one's own (out-group) are more resistant to change than corresponding associations to a member of one's own (in-group) (Olsson et al., 2005; Kubota et al., 2012), providing a possible link to discriminative behavior. Using a fear conditioning paradigm, we investigated the neural activity underlying aversive learning biases towards in-group (White) and out-group (Black) members, and their predictive value for discriminatory interactive behavior towards novel virtual members of the racial out-group (n=20). Our results indicate that activity in brain regions previously linked to conditioned fear and perception of individuals belonging to the racial out-groups, or otherwise stigmatized groups, jointly contribute to the expression of race-based biases in learning and behavior. In particular, we found that the amygdala and anterior insula (AI) played key roles in differentiating between in-group and out-group faces both when the faces were paired with an aversive event (acquisition) and when no more shocks were administered (extinction). In addition, functional connectivity between the amygdala and the fusiform gyrus increased during perception of conditioned out-group faces. Moreover, we showed that brain activity in the fear-learning-bias network was related to participants' discriminatory interactions with novel out-group members on a later day. Our findings are the first to identify the neural mechanism of fear learning biases towards out-group members, and its relationship to interactive behavior. Our findings provide important clues towards understanding the mechanisms underlying biases between social groups.

Sex differences in conditioned stimulus discrimination during context-dependent fear learning and its retrieval in humans: the role of biological sex, contraceptives and menstrual cycle phases.

BACKGROUND: Anxiety disorders are more prevalent in women than in men. Despite this sexual dimorphism, most experimental studies are conducted in male participants and studies focusing on sex differences are sparse. In addition, the role of hormonal contraceptives and menstrual cycle phase in fear conditioning and extinction processes remain largely unknown. METHODS: We investigated sex differences in context-dependent fear acquisition and extinction (day 1) and their retrieval/expression (day 2). Skin conductance responses (SCRs), fear and unconditioned stimulus expectancy ratings were obtained. RESULTS: We included 377 individuals (261 women) in our study. Robust sex differences were observed in all dependent measures. Women generally displayed higher subjective ratings but smaller SCRs than men and showed reduced excitatory/inhibitory conditioned stimulus (CS+/CS-) discrimination in all dependent measures. Furthermore, women using hormonal contraceptives showed reduced SCR CS discrimination on day 2 than men and free-cycling women, while menstrual cycle phase had no effect. LIMITATIONS: Possible limitations include the simultaneous testing of up to 4 participants in cubicles, which might have introduced a social component, and not assessing postexperimental contingency awareness. CONCLUSION: The response pattern in women shows striking similarity to previously reported sex differences in patients with anxiety. Our results suggest that pronounced deficits in associative discrimination learning and subjective expression of safety information (CS- responses) might underlie higher prevalence and higher symptom rates seen in women with anxiety disorders. The data call for consideration of biological sex and hormonal contraceptive use in future studies and may suggest that targeting inhibitory learning during therapy might aid precision medicine.

A review on human reinstatement studies: an overview and methodological challenges.

In human research, studies of return of fear (ROF) phenomena, and reinstatement in particular, began only a decade ago and recently are more widely used, e.g., as outcome measures for fear/extinction memory manipulations (e.g., reconsolidation). As reinstatement research in humans is still in its infancy, providing an overview of its stability and boundary conditions and summarizing methodological challenges is timely to foster fruitful future research. As a translational endeavor, clarifying the circumstances under which (experimental) reinstatement occurs may offer a first step toward understanding relapse as a clinical phenomenon and pave the way for the development of new pharmacological or behavioral ways to prevent ROF. The current state of research does not yet allow pinpointing these circumstances in detail and we hope this review will aid the research field to advance in this direction. As an introduction, we begin with a synopsis of rodent work on reinstatement and theories that have been proposed to explain the findings. The review however mainly focuses on reinstatement in humans. We first describe details and variations of the experimental setup in reinstatement studies in humans and give a general overview of results. We continue with a compilation of possible experimental boundary conditions and end with the role of individual differences and behavioral and/or pharmacological manipulations. Furthermore, we compile important methodological and design details on the published studies in humans and end with open research questions and some important methodological and design recommendations as a guide for future research.

Observational threat learning influences costly avoidance behaviour in healthy humans.

Avoidance is an essential behaviour for ensuring safety in uncertain and dangerous environments. One way to learn what is dangerous and must be avoided is through observational threat learning. This online study explored the behavioural implications of observed threat learning, examining how participants avoided or approached a learned threat and how this affected their movement patterns. Participants (n = 89) completed an observational threat learning task, rating their fear, discomfort, and physical arousal in response to conditioned stimuli. The retrieval of learned threat was reassessed 24 h later, followed by a reminder of the observed threat associations. Participants subsequently completed a computerised avoidance task, in which they navigated from a starting point to an endpoint by selecting one of two doors, each associated with either safety or danger, relying on observed information. Opting for the safe door entailed increased effort to attain the goal. Results demonstrated that observational threat learning influenced avoidance behaviour and decision-making dependent on baseline effort level. Participants tended to exhibit thigmotaxis, staying close to walls and taking extra steps to reach their goal. This behaviour indirectly mediated the number of steps taken. This study provides valuable insights into avoidance behaviour following observational threat learning in healthy humans.

Nicotine reduces discrimination between threat and safety in the hippocampus, nucleus accumbens and amygdala.

Nicotine intake is linked to the maintenance and development of anxiety disorders and impairs adaptive discrimination of threat and safety in rodents and humans. Yet, it is unclear if nicotine exerts a causal pharmacological effect on the affective and neural mechanisms that underlie aversive learning. We conducted a pre-registered, pseudo-randomly and double-blinded pharmacological fMRI study to investigate the effect of acute nicotine on Fear Acquisition and Extinction in non-smokers (n = 88). Our results show that nicotine administration led to decreased discrimination between threat and safety in subjective fear. Nicotine furthermore decreased differential (threat vs. safety) activation in the hippocampus, which was functionally coupled with Nucleus Accumbens and amygdala, compared to placebo controls. Additionally, nicotine led to enhanced physiological arousal to learned threats and overactivation of the ventral tegmental area. This study provides mechanistic evidence that single doses of nicotine impair neural substrates of adaptive aversive learning in line with the risk for the development of pathological anxiety.

Learning by observing: a systematic exploration of modulatory factors and the impact of observationally induced placebo and nocebo effects on treatment outcomes.

Introduction: Observational learning (OL) refers to learning through observing other people's behavior. OL has been suggested as an effective and simple tool to evoke treatment expectations and corresponding placebo and nocebo effects. However, the exact mechanisms by which OL shapes treatment outcomes, its moderating factors and possible areas of application remain unclear. We thus reviewed the existing literature with two different literature searches to answer the following questions: Which influencing factors contribute to OL-induced placebo and nocebo effects (in healthy volunteers and patients) and how large are these effects (search 1)? In which medical fields has OL been used so far to modulate treatment expectancy and treatment outcomes in patients, their caregivers, and at-risk groups (search 2)? We also aimed to explore whether and how the assessment of treatment expectations has been incorporated. Methods: We conducted two independent and comprehensive systematic literature searches, both carried out on September 20, 2022. Results: We identified 21 studies that investigated OL-mediated placebo and nocebo effects for pain and itch, the (placebo) efficacy of sham treatment on anxiety, and the (nocebo) induction of medication side effects (search 1). Studies showed that OL can efficiently induce placebo and nocebo effects across different presentation modes, with medium effect sizes on average: placebo effects, d = 0.79 (range: d = -0.36-1.58), nocebo effects, d = 0.61 (range: d = 0.04-1.5). Although several moderating factors have been investigated, their contribution to OL-induced effects remains unclear because of inconsistent results. Treatment expectation was assessed in only four studies. Regarding medical applications of OL (search 2), we found 12 studies. They showed that OL was effectively applied in preventive, therapeutic and rehabilitative interventions and that it was mainly used in the field of psychosomatics. Discussion: OL effects on treatment outcomes can be both positive and negative. Future research should investigate which individuals would benefit most from OL and how OL can be implemented most effectively to induce placebo and avoid nocebo effects in clinical settings. Systematic review registration: This work was preregistered at the Center for Open Science as open-ended registration (doi: 10.17605/OSF.IO/FVHKE). The protocol can be found here: https://archive.org/details/osf-registrations-fvhke-v1.

Inferring danger with minimal aversive experience.

Learning about threats is crucial for survival and fundamentally rests upon Pavlovian conditioning. However, Pavlovian threat learning is largely limited to detecting known (or similar) threats and involves first-hand exposure to danger, which inevitably poses a risk of harm. We discuss how individuals leverage a rich repertoire of mnemonic processes that operate largely in safety and significantly expand our ability to recognize danger beyond Pavlovian threat associations. These processes result in complementary memories - acquired individually or through social interactions - that represent potential threats and the relational structure of our environment. The interplay between these memories allows danger to be inferred rather than directly learned, thereby flexibly protecting us from potential harm in novel situations despite minimal prior aversive experience.

Opioid antagonism in humans: a primer on optimal dose and timing for central mu-opioid receptor blockade.

Non-human animal studies outline precise mechanisms of central mu-opioid regulation of pain, stress, affiliation and reward processing. In humans, pharmacological blockade with non-selective opioid antagonists such as naloxone and naltrexone is typically used to assess involvement of the mu-opioid system in such processing. However, robust estimates of the opioid receptor blockade achieved by opioid antagonists are missing. Dose and timing schedules are highly variable and often based on single studies. Here, we provide a detailed analysis of central opioid receptor blockade after opioid antagonism based on existing positron emission tomography data. We also create models for estimating opioid receptor blockade with intravenous naloxone and oral naltrexone. We find that common doses of intravenous naloxone (0.10-0.15 mg/kg) and oral naltrexone (50 mg) are more than sufficient to produce full blockade of central MOR (>90% receptor occupancy) for the duration of a typical experimental session (~60 min), presumably due to initial super saturation of receptors. Simulations indicate that these doses also produce high KOR blockade (78-100%) and some DOR blockade (10% with naltrexone and 48-74% with naloxone). Lower doses (e.g., 0.01 mg/kg intravenous naloxone) are estimated to produce less DOR and KOR blockade while still achieving a high level of MOR blockade for ~30 min. The models and simulations form the basis of two novel web applications for detailed planning and evaluation of experiments with opioid antagonists. These tools and recommendations enable selection of appropriate antagonists, doses and assessment time points, and determination of the achieved receptor blockade in previous studies.