Heightened extended amygdala metabolism following threat characterizes the early phenotypic risk to develop anxiety-related psychopathology.

Children with an anxious temperament are prone to heightened shyness and behavioral inhibition (BI). When chronic and extreme, this anxious, inhibited phenotype is an important early-life risk factor for the development of anxiety disorders, depression and co-morbid substance abuse. Individuals with extreme anxious temperament often show persistent distress in the absence of immediate threat and this contextually inappropriate anxiety predicts future symptom development. Despite its clear clinical relevance, the neural circuitry governing the maladaptive persistence of anxiety remains unclear. Here, we used a well-established nonhuman primate model of childhood temperament and high-resolution 18fluorodeoxyglucose positron emission tomography (FDG-PET) imaging to understand the neural systems governing persistent anxiety and to clarify their relevance to early-life phenotypic risk. We focused on BI, a core component of anxious temperament, because it affords the moment-by-moment temporal resolution needed to assess contextually appropriate and inappropriate anxiety. From a pool of 109 peri-adolescent rhesus monkeys, we formed groups characterized by high or low levels of BI, as indexed by freezing in response to an unfamiliar human intruder's profile. The high-BI group showed consistently elevated signs of anxiety and wariness across >2 years of assessments. At the time of brain imaging, 1.5 years after initial phenotyping, the high-BI group showed persistently elevated freezing during a 30-min 'recovery' period following an encounter with the intruder-more than an order of magnitude greater than the low-BI group-and this was associated with increased metabolism in the bed nucleus of the stria terminalis, a key component of the central extended amygdala. These observations provide a neurobiological framework for understanding the early phenotypic risk to develop anxiety-related psychopathology, for accelerating the development of improved interventions, and for understanding the origins of childhood temperament.

Evolutionarily conserved prefrontal-amygdalar dysfunction in early-life anxiety.

Some individuals are endowed with a biology that renders them more reactive to novelty and potential threat. When extreme, this anxious temperament (AT) confers elevated risk for the development of anxiety, depression and substance abuse. These disorders are highly prevalent, debilitating and can be challenging to treat. The high-risk AT phenotype is expressed similarly in children and young monkeys and mechanistic work demonstrates that the central (Ce) nucleus of the amygdala is an important substrate. Although it is widely believed that the flow of information across the structural network connecting the Ce nucleus to other brain regions underlies primates' capacity for flexibly regulating anxiety, the functional architecture of this network has remained poorly understood. Here we used functional magnetic resonance imaging (fMRI) in anesthetized young monkeys and quietly resting children with anxiety disorders to identify an evolutionarily conserved pattern of functional connectivity relevant to early-life anxiety. Across primate species and levels of awareness, reduced functional connectivity between the dorsolateral prefrontal cortex, a region thought to play a central role in the control of cognition and emotion, and the Ce nucleus was associated with increased anxiety assessed outside the scanner. Importantly, high-resolution 18-fluorodeoxyglucose positron emission tomography imaging provided evidence that elevated Ce nucleus metabolism statistically mediates the association between prefrontal-amygdalar connectivity and elevated anxiety. These results provide new clues about the brain network underlying extreme early-life anxiety and set the stage for mechanistic work aimed at developing improved interventions for pediatric anxiety.

CRHR1 genotypes, neural circuits and the diathesis for anxiety and depression.

The corticotrophin-releasing hormone (CRH) system integrates the stress response and is associated with stress-related psychopathology. Previous reports have identified interactions between childhood trauma and sequence variation in the CRH receptor 1 gene (CRHR1) that increase risk for affective disorders. However, the underlying mechanisms that connect variation in CRHR1 to psychopathology are unknown. To explore potential mechanisms, we used a validated rhesus macaque model to investigate association between genetic variation in CRHR1, anxious temperament (AT) and brain metabolic activity. In young rhesus monkeys, AT is analogous to the childhood risk phenotype that predicts the development of human anxiety and depressive disorders. Regional brain metabolism was assessed with (18)F-labeled fluoro-2-deoxyglucose (FDG) positron emission tomography in 236 young, normally reared macaques that were also characterized for AT. We show that single nucleotide polymorphisms (SNPs) affecting exon 6 of CRHR1 influence both AT and metabolic activity in the anterior hippocampus and amygdala, components of the neural circuit underlying AT. We also find evidence for association between SNPs in CRHR1 and metabolism in the intraparietal sulcus and precuneus. These translational data suggest that genetic variation in CRHR1 affects the risk for affective disorders by influencing the function of the neural circuit underlying AT and that differences in gene expression or the protein sequence involving exon 6 may be important. These results suggest that variation in CRHR1 may influence brain function before any childhood adversity and may be a diathesis for the interaction between CRHR1 genotypes and childhood trauma reported to affect human psychopathology.

Incidental Findings from 16,400 Brain MRI Examinations of Research Volunteers.

BACKGROUND AND PURPOSE: Incidental findings are discovered in neuroimaging research, ranging from trivial to life-threatening. We describe the prevalence and characteristics of incidental findings from 16,400 research brain MRIs, comparing spontaneous detection by nonradiology scanning staff versus formal neuroradiologist interpretation. MATERIALS AND METHODS: We prospectively collected 16,400 brain MRIs (7782 males, 8618 females; younger than 1 to 94 years of age; median age, 38 years) under an institutional review board directive intended to identify clinically relevant incidental findings. The study population included 13,150 presumed healthy volunteers and 3250 individuals with known neurologic diagnoses. Scanning staff were asked to flag concerning imaging findings seen during the scan session, and neuroradiologists produced structured reports after reviewing every scan. RESULTS: Neuroradiologists reported 13,593/16,400 (83%) scans as having normal findings, 2193/16,400 (13.3%) with abnormal findings without follow-up recommended, and 614/16,400 (3.7%) with "abnormal findings with follow-up recommended." The most common abnormalities prompting follow-up were vascular (263/614, 43%), neoplastic (130/614, 21%), and congenital (92/614, 15%). Volunteers older than 65 years of age were significantly more likely to have scans with abnormal findings (P < .001); however, among all volunteers with incidental findings, those younger than 65 years of age were more likely to be recommended for follow-up. Nonradiologists flagged <1% of MRIs containing at least 1 abnormality reported by the neuroradiologists to be concerning enough to warrant further evaluation. CONCLUSIONS: Four percent of individuals who undergo research brain MRIs have an incidental, potentially clinically significant finding. Routine neuroradiologist review of all scans yields a much higher rate of significant lesion detection than selective referral from nonradiologists who perform the examinations. Workflow and scan review processes need to be carefully considered when designing research protocols.

Evaluation of the Diagnostic Performance of EU-TIRADS in Discriminating Benign from Malignant Thyroid Nodules: A Prospective Study in One Referral Center.

BACKGROUND: Several thyroid societies have endorsed ultrasound (US) malignancy risk stratification systems for thyroid nodules and the recently released European Thyroid Imaging Reporting and Data System (EU-TIRADS) needs large prospective studies for validation. OBJECTIVE: The purpose of our study was to evaluate the performance of EU-TIRADS in identifying thyroid nodules for fine-needle aspiration biopsy (FNAB) and its ability to reduce the number of unnecessary biopsies. METHODS: This was a single-center prospective study. From August 2017 to September 2018, 783 consecutive patients with 1,000 thyroid nodules underwent US examination and US-guided FNAB. A total of 741 patients (median age 50 years; range, 15-87 years; 649 females, 92 males) with 942 nodules (median largest diameter 14 mm; range, 4-96 mm) met the following inclusion criteria: (1) nodules with benign or malignant cytology - categories II and VI of the Bethesda System for Reporting Thyroid Cytopathology (BSRTC); (2) nodules with non-diagnostic and indeterminate cytology (BSRTC I, BSRTC III, and BSRTC IV), or suspicious for malignancy (BSRTC V), if postoperative histology was present; (3) nodules classified as BSRTC I and BSRTC III with a repeat FNAB and conclusive cytology. RESULTS: Of 942 nodules, 839 (89.1%) were benign and 103 (10.9%) were malignant. Nodules were classified as follows: EU-TIRADS 2 - 4.8%, EU-TIRADS 3 - 37.4%, EU-TIRADS 4 - 25.2%, and EU-TIRADS 5 - 32.6%. The malignancy rate in categories 2 to 5 was 0, 0, 3.8, and 30.6%, respectively. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of EU-TIRADS with a cut-off set at category 5 were 91.3, 74.6, 30.6, 98.6, and 76.4%, respectively. Diagnostic performance other than sensitivity and NPV was superior in nodules ≥10 mm. FNAB number would be reduced by 53.4% if FNAB criteria were strictly applied. When the indication for FNAB was applied as test positivity, the estimated sensitivity, specificity, PPV, and NPV of EU-TIRADS were 69.9, 56.3, 16.4, and 93.8%, respectively. CONCLUSION: EU-TIRADS provides effective malignancy risk stratification that can guide the selection of thyroid nodules for biopsy. The application of the guidelines criteria for FNAB in the clinical practice might reduce significantly the number of unnecessary FNAB.

Thyroglossal duct cyst carcinoma.

Not required for Clinical Vignette.

Serotonin transporter binding and genotype in the nonhuman primate brain using [C-11]DASB PET.

UNLABELLED: The length polymorphism of the serotonin (5-HT) transporter gene promoter region has been implicated in altered 5-HT function and, in turn, neuropsychiatric illnesses, such as anxiety and depression. The nonhuman primate has been used as a model to study anxiety-related mechanisms in humans based upon similarities in behavior and the presence of a similar 5-HT transporter gene polymorphism. Stressful and threatening contexts in the nonhuman primate model have revealed 5-HT transporter genotype dependent differences in regional glucose metabolism. Using the rhesus monkey, we examined the extent to which serotonin transporter genotype is associated with 5-HT transporter binding in brain regions implicated in emotion-related pathology. METHODS: Genotype data and high resolution PET scans were acquired in 29 rhesus (Macaca mulatta) monkeys. [C-11]DASB dynamic PET scans were acquired for 90 min in the anesthetized animals and images of distribution volume ratio (DVR) were created to serve as a metric of 5-HT transporter binding for group comparison based on a reference region method of analysis. Regional and voxelwise statistical analysis were performed with corrections for anatomical differences in gray matter probability, sex, age and radioligand mass. RESULTS: There were no significant differences when comparing l/l homozygotes with s-carriers in the regions of the brain implicated in anxiety and mood related illnesses (amygdala, striatum, thalamus, raphe nuclei, temporal and prefrontal cortex). There was a significant sex difference in 5-HT transporter binding in all regions with females having 18%-28% higher DVR than males. CONCLUSIONS: Because these findings are consistent with similar genotype findings in humans, this further strengthens the use of the rhesus model for studying anxiety-related neuropathologies.

The serotonin transporter genotype is associated with intermediate brain phenotypes that depend on the context of eliciting stressor.

A variant allele in the promoter region of the serotonin transporter gene, SLC6A4, the s allele, is associated with increased vulnerability to develop anxiety-related traits and depression. Furthermore, functional magnetic resonance imaging (fMRI) studies reveal that s carriers have increased amygdala reactivity in response to aversive stimuli, which is thought to be an intermediate phenotype mediating the influences of the s allele on emotionality. We used high-resolution microPET [18F]fluoro-2-deoxy-D-glucose (FDG) scanning to assess regional brain metabolic activity in rhesus monkeys to further explore s allele-related intermediate phenotypes. Rhesus monkeys provide an excellent model to understand mechanisms underlying human anxiety, and FDG microPET allows for the assessment of brain activity associated with naturalistic environments outside the scanner. During FDG uptake, monkeys were exposed to different ethologically relevant stressful situations (relocation and threat) as well as to the less stressful familiar environment of their home cage. The s carriers displayed increased orbitofrontal cortex activity in response to both relocation and threat. However, during relocation they displayed increased amygdala reactivity and in response to threat they displayed increased reactivity of the bed nucleus of the stria terminalis. No increase in the activity of any of these regions occurred when the animals were administered FDG in their home cages. These findings demonstrate context-dependent intermediate phenotypes in s carriers that provide a framework for understanding the mechanisms underlying the vulnerabilities of s-allele carriers exposed to different types of stressors.

Defensive behaviors in infant rhesus monkeys: environmental cues and neurochemical regulation.

To survive, primates must detect danger in time to activate appropriate defensive behaviors. In this study, the defensive behaviors of infant rhesus monkeys exposed to humans were characterized. It was observed that the direction of the human's gaze is a potent cue for the infant. Infants separated from their mothers were active and emitted frequent distress vocalizations. When a human entered the room but did not look at the infant, it became silent and froze in one position. If the human stared at the infant, it responded with aggressive barking. Alterations of the opiate system affected the frequency of the infant's distress calls without affecting barking and freezing, whereas benzodiazepine administration selectively reduced barking and freezing. This suggests that opiate and benzodiazepine systems regulate specific defensive behaviors in primates and that these systems work together to mediate behavioral responses important for survival.

Dexamethasone fails to suppress beta-endorphin plasma concentrations in humans and rhesus monkeys.

In humans and rhesus monkeys, dexamethasone decreased concentrations of plasma cortisol but did not alter circulating beta-endorphin immunoreactivity. Contrary to current theory suggesting that pituitary beta-endorphin and adrenocorticotropic hormone are controlled by identical regulatory mechanisms for synthesis and release, our evidence suggests that in higher primates the established glucocorticoid feedback mechanism for the adrenocorticotropic hormone-cortisol system does not regulate beta-endorphin secretion in the same way.

Mood and behavioral effects of physostigmine on humans are accompanied by elevations in plasma beta-endorphin and cortisol.

Administration of physostigmine to normal volunteers produced significant elevations in plasma cortisol and beta-endorphin immunoreactivity as well as alterations in mood, cognition, and behavior. These observations might be explained by a cholinergically mediated stress syndrome. However, peak elevations in plasma beta-endorphin immunoreactivity (but not in plasma cortisol) were significantly correlated with physostigmine-induced increases in depression ratings. These results suggest that a cholinergically mediated beta-endorphin pathway may be involved in the observed affective changes.

DeltaF508 CFTR protein expression in tissues from patients with cystic fibrosis.

Heterologous expression of the cystic fibrosis transmembrane conductance regulator (CFTR) provided evidence that the major cystic fibrosis (CF) mutation DeltaF508 leads to defective protein folding in the endoplasmic reticulum, which prevents its processing and targeting to the cell surface. In this study, we investigated endogenous CFTR expression in skin biopsies and respiratory and intestinal tissue specimens from DeltaF508 homozygous and non-CF patients, using immunohistochemical and immunoblot analyses with a panel of CFTR antibodies. CFTR expression was detected at the luminal surface of reabsorptive sweat ducts and airway submucosal glands, at the apex of ciliated cells in pseudostratified respiratory epithelia and of isolated cells of the villi of duodenum and jejunum, and within intracellular compartments of intestinal goblet cells. In DeltaF508 homozygous patients, expression of the mutant protein proved to be tissue specific. Whereas DeltaF508 CFTR was undetectable in sweat glands, the expression in the respiratory and intestinal tracts could not be distinguished from the wild-type by signal intensity or localization. The tissue-specific variation of DeltaF508 CFTR expression from null to apparently normal amounts indicates that DeltaF508 CFTR maturation can be modulated and suggests that determinants other than CFTR mislocalization should play a role in DeltaF508 CF respiratory and intestinal disease.

CFTR and differentiation markers expression in non-CF and delta F 508 homozygous CF nasal epithelium.

Human nasal polyps from non-CF and delta F 508 homozygous CF patients were used to compare the expression of CFTR and markers epithelial differentiation, such as cytokeratins (CK) and desmoplakins (DP), at the transcriptional and translational levels. mRNA expression was assessed by semiquantitative RT/PCR kinetic assays while the expression and distribution of proteins were evaluated by immunofluorescence analysis. In parallel, for each nasal tissue specimen, the importance of surface epithelium remodeling and inflammation was estimated after histological observations. Our results show that the steady-state levels of CFTR, CK13, CK18, CK18, CK14, or DP 1 mRNA transcripts in delta F 508 CF nasal polyps were not significantly different from those of non-CF tissues. A variability in the CFTR mRNA transcript level and in the pattern of CFTR immunolabeling has been observed between the different tissue samples. However, no relationship was found between the level of CFTR mRNA transcripts and the CFTR protein expression and distribution, either in the non-CF or in the CF group. The histological observations of non-CF and CF nasal polyp tissue indicated that the huge variations in the expression and distribution of the CFTR protein were associated with the variations in the degree of surface epithelium remodeling and inflammation in the lamina propria. A surface epithelium, showing a slight basal cell hyperplasia phenotype associated with diffuse inflammation, was mainly characterized by a CFTR protein distribution at the apex of ciliated cells in both non-CF and CF specimens. In contrast, in a remodeled surface epithelium associated with severe inflammation, CFTR protein presented either a diffuse distribution in the cytoplasm of ciliated cells, or was absent. These results suggest that abnormal expression and distribution of the CFTR protein of CF airways is not only caused by CFTR mutations. Airway surface epithelium remodeling and inflammation could play a critical role in the posttranscriptional and/or the posttranslational regulation of the CFTR protein expression in non-CF and CF airways.

Changes in Serum Creatinine Concentration and Acute Kidney Injury (AKI) Grade in Dogs Treated with Hydroxyethyl Starch 130/0.4 From 2013 to 2015.

BACKGROUND: Hydroxyethyl starch (HES) solutions may cause acute kidney injury (AKI) in humans. OBJECTIVE: To compare AKI grades in 94 dogs exposed and 90 dogs that were unexposed to 6% HES-130/0.4. ANIMALS: Dogs receiving 6% HES-130/0.4 (HES cohort) or crystalloids (unexposed cohort) between 2013 and 2015. METHODS: Historical cohort study. Diagnosis, total cumulative dose and total mL/kg of HES administered, time frame of HES administration and serum creatinine concentrations up to 90 days after initiation of HES treatment were retrospectively reviewed. The AKI grades were retrospectively determined by IRIS guidelines. RESULTS: Exposed dogs received a median cumulative dose of 69.4 mL/kg (range, 2-429 mL/kg) HES over a median of 4 (range, 1-16) days, resulting in a median dose of 20.7 (range, 2-87) mL/kg/d. Although the cohorts differed in terms of age and diagnosis, AKI grades were not significantly different at the evaluated short- and long-term time points. Results of ordinal logistic regression identified the number of days of HES administration as significantly associated with an increase in AKI grade within 10 days (P = .038), whereas there was no significant association among HES exposure, HES mL/kg/d, and an increase in AKI grade. CONCLUSIONS AND CLINICAL IMPORTANCE: HES-130/0.4-treated dogs were not more prone to develop AKI than HES-untreated, but the number of HES days was significantly associated with an increase in AKI grade within 10 days post-HES administration. The time frame of HES treatment should be kept short. Prospective, randomized clinical trials are required to assess the effect of HES on renal function in dogs.

Effects of Hydroxyethyl Starch 130/0.4 on Serum Creatinine Concentration and Development of Acute Kidney Injury in Nonazotemic Cats.

BACKGROUND: Hydroxyethyl-starch (HES) solutions might have renal adverse effects in humans and dogs. OBJECTIVE: To determine if administration of 6% HES-130/0.4 is associated with an increase in serum creatinine concentration and development of acute kidney injury (AKI) in nonazotemic cats. ANIMALS: A total of 62 critically ill cats; 26 HES exposed and 36 unexposed. METHODS: Retrospective cohort study (2012-2015). Serum creatinine concentrations were recorded and changes in serum creatinine concentrations before exposure (baseline) and 2-10 and 11-90 days, respectively, were determined. Development of AKI was defined as a > 150% increase or >26 µmol/L increase in serum creatinine concentration from baseline. Risk factors, such as HES administration, cumulative volume of HES (mL/kg) and number of days of HES administration leading to development of AKI, and change in serum creatinine were analyzed. RESULTS: Cats in the HES cohort received a mean volume of 98.5 ± 76.2 mL/kg (range, 8-278 mL/kg) HES over a median of 4 (range, 1-11) days, resulting in a median dose of 20.1 (range, 8-40.5) mL/kg per day. Short-term %change in serum creatinine concentration (P = 0.40) and development of AKI (P = 0.32) were not significantly different between cohorts. Multivariable logistic regression did not identify HES dose in mL/kg (P = 0.33) and number of days of HES application (P = 0.49) as a risk factor for development of AKI. CONCLUSION AND CLINICAL IMPORTANCE: Hydroxyethyl-starch administration to critically ill nonazotemic cats seems to be safe. A larger prospective study is required to determine the effect of HES administration at higher dosages and for prolonged time periods.

The primate amygdala mediates acute fear but not the behavioral and physiological components of anxious temperament.

Temperamentally anxious individuals can be identified in childhood and are at risk to develop anxiety and depressive disorders. In addition, these individuals tend to have extreme asymmetric right prefrontal brain activity. Although common and clinically important, little is known about the pathophysiology of anxious temperament. Regardless, indirect evidence from rodent studies and difficult to interpret primate studies is used to support the hypothesis that the amygdala plays a central role. In previous studies using rhesus monkeys, we characterized an anxious temperament endophenotype that is associated with excessive anxiety and fear-related responses and increased electrical activity in right frontal brain regions. To examine the role of the amygdala in mediating this endophenotype and other fearful responses, we prepared monkeys with selective fiber sparing ibotenic acid lesions of the amygdala. Unconditioned trait-like anxiety-fear responses remained intact in monkeys with >95% bilateral amygdala destruction. In addition, the lesions did not affect EEG frontal asymmetry. However, acute unconditioned fear responses, such as those elicited by exposure to a snake and to an unfamiliar threatening conspecific were blunted in monkeys with >70% lesions. These findings demonstrate that the primate amygdala is involved in mediating some acute unconditioned fear responses but challenge the notion that the amygdala is the key structure underlying the dispositional behavioral and physiological characteristics of anxious temperament.

Diurnal variation in cerebrospinal fluid prolactin concentration of the rhesus monkey.

PRL immunoreactivity in the cerebrospinal fluid (CSF) of the rhesus monkey was found found to undergo a diurnal variation. Hourly samples of CSF from four adult male rhesus monkeys showed a mean peak PRL immunoreactivity of 6.30 +/- 2.53 ng/ml at 0200 h and a mean minimum concentration at 1500 h of 3.15 +/- .68 ng/ml. To our knowledge, this is the first report of a significant diurnal variation in the concentration of an anterior pituitary peptide hormone in CSF.

ACTH in plasma and CSF in the rhesus monkey.

Adrenocorticotrophic hormone (ACTH) and other biosynthetically related peptides are found both in the brain and peripherally, but the function and regulation of these substances differ in the brain and in the periphery. It has been suggested that measurement of peptide hormones in the cerebrospinal fluid (CSF) might provide information relevant to the diagnosis and pathophysiology of neurological and psychiatric illnesses. We report experiments using a rhesus monkey model to evaluate parameters affecting CSF ACTH concentrations. We found that CSF ACTH concentrations follow a diurnal rhythm that is markedly different from that in plasma, concentrations of ACTH in monkey CSF, but not in plasma, increased significantly after 4 days of social separation, and CSF ACTH concentrations did not change after hypophysectomy. These results suggest that CSF ACTH concentrations reflect the activity of brain and not peripheral ACTH systems.

Corticotropin-releasing hormone and animal models of anxiety: gene-environment interactions.

The study of the neural substrates underlying stress and anxiety has in recent years been enriched by a burgeoning pool of genetic information gathered from rodent studies. Two general approaches have been used to characterize the interaction of genetic and environmental factors in stress regulation: the evaluation of stress-related behavioral and endocrine responses in animals with targeted deletion or overexpression of specific genes and the evaluation of changes in central nervous system gene expression in response to environmental perturbations. We review recent studies that have used molecular biology and genetic engineering techniques such as in situ hybridization, transgenic animal, and antisense oligonucleotide gene-targeting methodologies to characterize the function of corticotropin-releasing hormone (CRH) system genes in stress. The effects of genetic manipulations of each element of the CRH system (CRH, its two receptors, and its binding protein) on stress-related responses are summarized. In addition, the effects of stress (acute, repeated, or developmental) on CRH system gene expression are described. The results from these studies indicate that experimentally engineered or stress-induced dysregulation of gene expression within the CRH system is associated with aberrant responses to environmental contingencies. These results are discussed in the context of how CRH system dysfunction might contribute to stress-related psychopathology and are presented in conjunction with clinical findings of CRH system dysregulation in psychiatric illness. Finally, future research strategies (i.e., high-throughput gene screening and novel gene-targeting methodologies) that may be used to gain a fuller understanding of how CRH system gene expression affects stress-related functioning are discussed.