Stimuli presentation and data collection used E-Prime software

Our results indicate that screening, along with brief interventions and referrals to treatment, may be particularly important for identifying and providing early intervention for women of reproductive age prior to conception who may be at greater risk for prenatal alcohol and nicotine use. It is important that these conversations are supportive and non-punitive, focused on providing education and support to help women make informed decisions about substance use to increase the likelihood of future substance free pregnancies. Women’s health clinicians should also discuss risks associated with prenatal substance use at prenatal intake appointments to ensure that all patients receive the recommendation for complete abstinence throughout the pregnancy. Further, education about tracking one’s menstrual cycle for earlier recognition of pregnancy could potentially help women stop alcohol or nicotine use earlier, particularly in cases where women are not actively trying to conceive. Future studies that examine pregnancy intentions may be useful to understand whether trends in prenatal alcohol and nicotine use vary among women whose pregnancies are intended versus unintended. In contrast to declines in the prevalence and frequency of alcohol and nicotine use among pregnant women seen in the current study, recent studies have found increases in the frequency and prevalence of cannabis use during pregnancy . Cannabis use during pregnancy commonly co-occurs with alcohol and nicotine use , and additional studies are needed to better understand patterns of co-use of alcohol, nicotine and cannabis among pregnant women over time.This study has a number of strengths,vertical cannabis grow including a large sample of diverse pregnant women universally screened for alcohol and nicotine use as part of standard prenatal care, data on self-reported frequency of use both in the year before pregnancy and during pregnancy, and repeated cross-sectional data spanning nine years.

There are also several study limitations. Our sample was limited to pregnant women who completed the self-reported substance use screening questionnaire as part of standard prenatal care. Findings may not be generalizable to pregnant women who did not complete the self-reported substance use screening questionnaire or to those who do not receive prenatal care, who may be more likely to use substances during pregnancy. Data on self reported alcohol and nicotine use came from the initial prenatal visit , and do not reflect continued use throughout pregnancy. We were unable to differentiate alcohol and nicotine use in pregnancy that occurred before versus after women realized they were pregnant, and many women in our sample who used alcohol or nicotine while pregnancy may have stopped as soon as they became aware of their pregnancy. Finally, our study may underestimate both the prevalence and frequency of alcohol and nicotine use before and during pregnancy as women may choose not to disclose their use to their healthcare provider.Studies of how neurobiological systems are linked to the transdiagnostic endophenotypic and phenotypic expression of psychopathology are particularly relevant to trauma-related psychopathology, as three of the most common trauma-related disorders—post traumatic stress disorder , major depressive disorder , and generalized anxiety disorder —are highly comorbid and share common transdiagnostic dimensions of threat and loss symptomatology . Trauma-related threat symptomatology includes intrusive thoughts and memories, and hyperarousal symptoms such as sleep disturbance and hypervigilance, whereas trauma-related loss symptomatology includes emotional numbing and depressive/dysphoric and generalized anxiety symptoms. Elucidation of neurobiological systems implicated in trauma-related endophenotypes can inform etiologic models of traumarelated psychopathology, as well as the development of more targeted, mechanism-based prevention and treatment strategies.

Attentional bias to threat is one of the core endophenotypic characteristics of trauma-related psychopathology . Attentional biases to threatening information, such as faces and words, which are often assessed using a dot-probe paradigm, have been found to contribute to and maintain the persistence of trauma-related threat symptomatology, even months to years after trauma exposure . Greater attentional bias to threat is also associated with exaggerated fear expression and impaired extinction in individuals with PTSD .Hyperarousal symptoms, such as exaggerated startle response during fear learning, in particular, have been found to contribute to attentional bias to threat in symptomatic trauma survivors . Recent functional neuroimaging work has implicated increased amygdala activation in relation to attentional bias to threat among individuals with PTSD , suggesting that the amygdala modulates the orientation of attention toward and processing of threatening information in this population. Although cannabinoid type 1 receptors are widely distributed in the human brain , they are found in particularly high concentrations in the amygdala, and have been associated with the processing and storage of threat-related memories, as well as the coordination of threat-related behaviors . Recently, we reported in vivo evidence of abnormal CB1 receptor-mediated endocannabinoid signaling in individuals with PTSD and suggested that increased CB1 receptor availability may be a molecular adaptation to reduced endocannabinoid availability. In addition to this work, a large body of preclinical studies has found strong support for a major role of the endocannabinoid anandamide and CB1 receptor signaling in the amygdala in modulating stress-induced threat behaviors . Understanding how key neuroreceptor systems such as CB1 relate to intermediate endophenotypic and phenotypic expression of trauma-related psychopathology may thus provide insight into molecular targets that could inform the development of mechanism-based treatment approaches. To date, however, human data evaluating this possibility are lacking.

In the current study, we aimed to address this gap in the literature by using the CB1 receptor antagonist radiotracer [11C]OMAR, which measures volume of distribution linearly related to CB1 receptor availability, to evaluate the relationship between CB1 receptor availability in the amygdala, and objectively assessed attentional bias to threat, and the transdiagnostic and dimensional expression of trauma-related threat and loss symptomatology. To obtain a sample that encompassed the full-dimensional range of study measures , we employed an inclusive sampling approach by recruiting a sample of individuals who represented a broad transdiagnostic and dimensional spectrum of trauma-related psychopathology, ranging from healthy, nontrauma-exposed individuals to trauma-exposed individuals with severe trauma-related psychopathology. On the basis of prior work linking CB1 receptor availability in the amygdala to threat processing and threat symptomatology to attentional bias to threat , we hypothesized that greater CB1 receptor availability in the amygdala would be associated with greater attentional bias to threat,vertical grow light as well as increased severity of threat symptomatology, particularly hyperarousal. We then evaluated a mediational model to examine whether attentional bias to threat mediated the relationship between CB1 receptor availability in the amygdala and trauma-related psychopathology.Lifetime traumatic events were assessed using the Traumatic Life Events Questionnaire and psychiatric diagnoses were established using DSM-IV-TR criteria and the Structured Clinical Interview for DSM-IV that was administered by an experienced master- or doctoral level psychiatric clinician. Only traumatic events that met criteria A1 and A2 for a DSM-IV-TR-based diagnosis of PTSD were counted toward participants’ trauma histories in this study. Nontrauma-exposed healthy adults did not report any trauma exposures on the TLEQ and did not have any lifetime psychiatric diagnosis, including substance abuse or dependence or nicotine dependence. Severity of trauma-related threat and loss symptomatology was assessed using the Clinician-Administered PTSD Scale for DSM-IV ; the Hamilton Rating Scale for Depression to assess depressive symptoms; and the Hamilton Rating Scale for Anxiety to assess nonspecific anxiety symptoms. Scores on these structured clinician-administered measures of trauma-related psychopathology represented a transdiagnostic and dimensional spectrum of trauma-related psychopathology, ranging from nontrauma-exposed asymptomatic adults to trauma exposed adults with severe trauma-related psychopathology . All participants were evaluated by physical examination, electrocardiogram, standard blood chemistry, hematology laboratory testing, toxicology testing, and urinalysis. All but two participants were psychotropic medication naive, and two took antidepressants for less than a week before the study but were medication free for at least 6 months before the study. Participants with significant medical or neurologic conditions, with substance abuse within 12 months of the scan, lifetime history of intravenous substance dependence, or with history of head injury that involved loss of consciousness were excluded from the study. Lifetime cannabis abuse/dependence was an exclusion criterion, and occasional cannabis users were eligible to participate but not if they had used cannabis within 12 months of the scan. The absence of substance use was determined by self-report and confirmed by the results of urine toxicology and breathalyzer tests at screening and on the days when magnetic resonance imaging and positron emission tomography scans were conducted.

The medical and psychiatric evaluation was followed by MRI and a resting state PET scan on a High Resolution Research Tomograph PET scanner with the CB1-selective radio ligand [11C]OMAR . To obtain plasma anandamide levels, blood samples were collected at the time of tracer injection and processed immediately after collection in the laboratory that is adjacent to the scan room and frozen at 80 1C until analyzed, as previously described .The dot-probe task was composed of 160 trials. Each trial started with a fixation cross presented in the center of the screen for 500 ms. When the fixation cross disappeared, two words in 12-pt Arial font immediately appeared in the center of the screen for 500 ms, one above and one below the location of the fixation cross, separated by 1.5 cm. Following the presentation of the words, a target probe appeared in the location previously occupied by one of the words. The probe remained on the screen until participants responded, after which the next trial started. Participants were instructed to focus their attention on the fixation cross at the start of each trial, and when a probe appeared they were to identify the probe letter using a designated mouse button, as quickly as possible. Given the heterogeneity of trauma histories in our sample, the stimuli used were 32 trauma-related and 64 neutral words that were selected from a larger list developed by MacLeod et al . Word pairs were chosen for salience to the experience of traumatic life events . Word pairs were matched in terms of first letter, number of letters, and frequency of usage in the English language, as suggested by MacLeod et al , and were presented in random order. To reduce the effect of anticipatory responding and outliers, response times o200 ms and 43 SD above the mean for each trial were discarded . Attentional bias to threat was calculated as the difference between average RT to targets at neutral word locations and average RT to targets at threat word locations. Negative scores indicate attentional bias away from threat, whereas positive scores indicate attentional bias toward threat.Simple descriptive statistics were computed to summarize demographic, trauma-related, and clinical variables for the sample. To reduce symptom clusters into composite measures of trauma-related threat and loss symptomatology based on prior work , we conducted two principal components analyses : the first contained CAPS measures of re-experiencing and hyperarousal symptoms , and the second contained CAPS measure of avoidance/numbing symptoms and HAM-D and HAM-A measures of major depressive and anxiety symptoms. Pearson or Spearman correlations, as appropriate based on data distributions, were then computed to evaluate associations between [11C]OMAR VT values in the amygdala, attentional bias to threat, and composite measures of trauma-related threat and loss symptomatology. If significant associations were observed, exploratory post hoc analyses were conducted to evaluate associations between component aspects of composite measures; exploratory post hoc analyses were also conducted to evaluate associations between [11C]OMAR VT values in brain regions other than the amygdala in relation to attentional bias to threat; a was set to 0.01 for all of these analyses to reduce the likelihood of type I error. To evaluate whether attentional bias to threat mediated the relation between CB1 receptor availability in the amygdala and the phenotypic expression of traumarelated psychopathology, we conducted a bootstrapped mediation analysis with 10 000 replicates using Mplus version 7.11. Model fit was assessed using w2 , comparative fit index , and standardized root mean square residual fit statistics; by convention, non-significant w2 values, CFI values Z0.90, and SRMR values o0.05 indicate a good fit to the data .Using the CB1 receptor radiotracer [11C]OMAR, we found that greater CB1 receptor availability in the amygdala was associated with increased attentional bias to threat, as well as increased severity of trauma-related threat symptomatology in humans presenting with a broad dimensional spectrum of trauma-related psychopathology.