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.

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.

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.

Lateralized effects of diazepam on frontal brain electrical asymmetries in rhesus monkeys.

A growing body of literature has documented the differential role of the frontal regions of the two cerebral hemispheres in certain positive and negative affective processes. This corpus of evidence has led to the hypothesis of a possible differential effect of diazepam on asymmetry of frontal activation. To examine this question, nine infant rhesus monkeys were tested on two occasions during which brain electrical activity was recorded from left and right frontal and parietal scalp regions. During one session, recordings were obtained under a baseline restraint condition and then after an injection of diazepam (1 mg/kg). In the other session, following the same baseline restraint condition, a vehicle injection was given. In response to diazepam, the animals showed an asymmetrical decrease in power in the 4-8 Hz frequency band, which was most pronounced in the left frontal region. No change in electroencephalogram (EEG) activity was observed in response to vehicle. Asymmetry in parietal EEG activity was also unchanged by diazepam. Diazepam also produced overall reductions in power across different frequency bands in both frontal and parietal regions. Good test-retest stability of EEG measures of activation asymmetry was also found between the two testing sessions separated by three months. The possible proximal cause of the asymmetrical change in frontal brain electrical activity in response to diazepam, as well as the implications of these findings for understanding the mechanism of action of benzodiazepines are discussed.

Cerebrospinal fluid corticotropin-releasing hormone levels are elevated in monkeys with patterns of brain activity associated with fearful temperament.

BACKGROUND: Asymmetric patterns of frontal brain activity and brain corticotropin-releasing hormone (CRH) systems have both been separately implicated in the processing of normal and abnormal emotional responses. Previous studies in rhesus monkeys demonstrated that individuals with extreme right frontal asymmetric brain electrical activity have high levels of trait-like fearful behavior and increased plasma cortisol concentrations. METHODS: In this study we assessed cerebrospinal fluid (CSF) CRH concentrations in monkeys with extreme left and extreme right frontal brain electrical activity. CSF was repeatedly collected at 4, 8, 14, 40, and 52 months of age. RESULTS: Monkeys with extreme right frontal brain activity had increased CSF CRH concentrations at all ages measured. In addition, individual differences in CSF CRH concentrations were stable from 4 to 52 months of age. CONCLUSIONS: These findings suggest that, in primates, the fearful endophenotype is characterized by increased fearful behavior, a specific pattern of frontal electrical activity, increased pituitary-adrenal activity, and increased activity of brain CRH systems. Data from other preclinical studies suggests that the increased brain CRH activity may underlie the behavioral and physiological characteristics of fearful endophenotype.

Effects of beta-carboline on fear-related behavioral and neurohormonal responses in infant rhesus monkeys.

We examined the effects of the inverse benzodiazepine agonist ethyl-beta-carboline-3-carboxylate (beta-CCE) on behavioral, hormonal, and neurochemical responses in infant rhesus monkeys exposed to fearful situations. Our paradigm elicits three distinct adaptive patterns of defensive behavior. From previous work, we hypothesized that behaviors induced by attachment bond disruption are predominantly mediated by opiate systems, whereas behaviors induced by the threat of attack are mediated by benzodiazepine systems. When beta-CCE (0, 125, 250, and 500 micrograms/kg) was administered immediately after maternal separation, the 500 micrograms/kg dose increased freezing and the 250 and 500 micrograms/kg doses reduced environmental exploration. Test conditions produced increased plasma ACTH and cortisol concentrations and increased cerebrospinal fluid (CSF) concentrations of MHPG and DOPAC; beta-CCE did not further affect these metabolites. A dose of 1000 micrograms/kg of beta-CCE increased CSF concentrations of DOPAC and MHPG in infants left with their mothers. During test conditions, it further increased CSF MHPG (but not DOPAC) concentrations, and reduced cooing while increasing freezing and barking and other hostile behaviors. Our results thus confirm that benzodiazepine systems mediate threat-related behaviors and suggest that coos, which were thought to predominantly reflect the degree of distress during separation, can be modulated by the infant's level of fear. beta-CCE also activated stress-related pituitary-adrenal hormonal systems and brain norepinephrine (NE) and dopamine (DA) systems. These effects occurred when animals remained undisturbed in their home cages with their mothers, suggesting that benzodiazepine receptors directly modulate brain NE and DA systems.

Consistent reversal of abnormal DSTs after different antidepressant therapies in a patient with dementia.

The authors present a longitudinal case study of a 57-year-old man whose initial symptoms consisted of depression and dementia associated with an abnormal dexamethasone suppression test (DST). Antidepressant therapy resulted in consistent reversal of the DST and partial remission of the patient's vegetative signs. Nevertheless, the patient's condition deteriorated over 36 months to end-stage dementia.

Behavioral and physiologic effects of CRH administered to infant primates undergoing maternal separation.

Infant rhesus monkeys briefly separated from their mothers emit frequent distress vocalizations and alter their activity levels. These behavioral alterations are accompanied by physiologic changes and activation of the hypothalamic-pituitary-adrenal axis. Because corticotropin-releasing hormone (CRH) is a major regulator of the pituitary-adrenal system and has been implicated as a mediator of other aspects of the stress response, we examined the effects of centrally and peripherally administered CRH on the infant monkey's separation response. Intracerebroventricular (ICV) doses less than 10 micrograms did not alter behavior; 10-micrograms doses inhibited behavior without affecting distress vocalizations. The behavioral inhibition did not appear to be due to nonspecific sedation and may be related to increased fearfulness. It was accompanied by increases in adrenocorticotropic hormone (ACTH) and cortisol and small but significant decreases in body temperature and mean arterial pressure. Within an individual animal, no relationship was seen between ICV CRH's effects on physiologic parameters and its effects on behavior. ICV CRH produced significant increases in plasma concentrations of CRH, suggesting the possibility that the effects of CRH were mediated through peripheral mechanisms. However, 10 micrograms of CRH administered intravenously (IV) had no effect on behavior, blood pressure, or body temperature, nor did it increase plasma concentrations of ACTH or cortisol beyond the expected separation-induced elevation. This is interesting because plasma levels of CRH after IV CRH were much greater than after ICV CRH. Our findings suggest that in infant rhesus monkeys undergoing maternal separation, brain CRH systems mediate behavioral inhibition and pituitary-adrenal activation.

Limbic-hypothalamic-pituitary-adrenal axis activity and ventricular-to-brain ratio studies in affective illness and schizophrenia.

Some investigators have speculated that structural brain alterations observed in some psychiatric patients might be related to increased limbic-hypothalamic-pituitary-adrenal axis (LHPA) activity. To explore this hypothesis, we prospectively studied 166 research volunteers (19 patients with research diagnostic criteria (RDC) major depression, 9 patients with RDC bipolar depression, 45 patients with RDC schizophrenia, and 94 RDC normal controls), examining the relationship between magnetic resonance image-determined ventricular-to-brain ratio (VBR) and indices of LHPA axis function (cerebrospinal fluid (CSF) corticotropin-releasing factor (CRF), CSF adrenocorticotropic hormone (ACTH), and 24-hour urinary-free cortisol secretion). We observed no significant differences in mean VBR among the three patient groups and the normal control volunteers. Of the indices of LHPA activity, only CSF CRF concentrations distinguished the four subject groups, with CSF CRF being significantly elevated in the more severely depressed major depression patients. Indices of LHPA activity were not significantly correlated with VBR in any of the three patient groups or in the normal volunteers. These preliminary results suggest that VBR is not highly associated with alterations in LHPA activity, at least as determined cross-sectionally. Further longitudinal studies with reference to diagnostic subtypes, severity, symptom profiles, and more specific neuroanatomic regions may allow the elucidation of possible relationships between LHPA pathology and structural brain alterations.