This review brings together recent research from molecular, neural circuit, animal model, and human studies to help understand the neurodevelopmental mechanisms underlying social anxiety disorder. Social anxiety disorder is common and debilitating, and it often leads to further psychopathology. Numerous studies have demonstrated that extremely behaviorally inhibited and temperamentally anxious young children are at marked risk of developing social anxiety disorder. Recent work in human and nonhuman primates has identified a distributed brain network that underlies early-life anxiety including the central nucleus of the amygdala, the anterior hippocampus, and the orbitofrontal cortex. Studies in nonhuman primates have demonstrated that alterations in this circuit are trait-like in that they are stable over time and across contexts. Notably, the components of this circuit are differentially influenced by heritable and environmental factors, and specific lesion studies have demonstrated a causal role for multiple components of the circuit. Molecular studies in rodents and primates point to disrupted neurodevelopmental and neuroplastic processes within critical components of the early-life dispositional anxiety neural circuit. The possibility of identifying an early-life at-risk phenotype, along with an understanding of its neurobiology, provides an unusual opportunity to conceptualize novel preventive intervention strategies aimed at reducing the suffering of anxious children and preventing them from developing further psychopathology.

Background

Anxious temperament (AT) is an early-life heritable trait that predisposes individuals to develop anxiety and depressive disorders. Our previous work in preadolescent children suggests alterations in the uncinate fasciculus (UF), the white matter tract that connects prefrontal with limbic regions, in boys with anxiety disorders. Here, using a nonhuman primate model of AT, we tested whether this sexually dimorphic finding is evolutionarily conserved and examined the extent to which heritable and environmental influences contribute to UF microstructure.

Methods

Diffusion tensor images were collected in 581 young rhesus monkeys (1.89 ± 0.77 years old; 43.9% female). Using tract-based analyses, we assessed the relationship among AT, UF microstructure (as measured with fractional anisotropy), and sex. Heritability of tract microstructure was determined using oligogenic linkage analysis of this large multigenerational pedigree.

Results

We predicted and found a negative relation between AT and UF fractional anisotropy in male but not female monkeys (AT × sex; p = .032, 1-tailed). Additionally, heritability analyses revealed that variation in UF fractional anisotropy was largely due to nonheritable factors (h² = 0.185, p = .077).

Conclusions

These results demonstrate a cross-species, male-specific relation between UF microstructure and anxiety and provide a potential substrate for anxiety-related prefrontal-limbic dysregulation. The heritability analyses point to the importance of environmental influences on UF microstructure, which could be important in mediating the nonheritable components of pathological anxiety. These findings have the potential to guide new treatment strategies for childhood anxiety disorders and further support the use of nonhuman primates as a translational model to discover mechanisms underlying the development of anxiety.

BACKGROUND: Evidence suggests that early life adversity is associated with maladaptive behaviors and is commonly an antecedent of stress-related psychopathology. This is particularly relevant to rearing in primate species as infant primates depend on prolonged, nurturant rearing by caregivers for normal development. To further understand the consequences of early life rearing adversity, and the relation among alterations in behavior, physiology and brain function, we assessed young monkeys that had experienced maternal separation followed by peer rearing with behavioral, endocrine and multimodal neuroimaging measures. METHODS: 50 young rhesus monkeys were studied, half of which were rejected by their mothers and peer reared, and the other half were reared by their mothers. Assessments were performed at approximately 1.8 years of age and included: threat related behavioral and cortisol responses, cerebrospinal fluid (CSF) measurements of oxytocin and corticotropin releasing hormone (CRH), and multimodal neuroimaging measures (anatomical scans, resting functional connectivity, diffusion tensor imaging, and threat-related regional glucose metabolism). RESULTS: The results demonstrated alterations across behavioral, endocrine, and neuroimaging measures in young monkeys that were reared without their mothers. At a behavioral level in response to a potential threat, peer reared animals engaged in significantly less freezing behavior (p = 0.022) along with increased self-directed behaviors (p < 0.012). Levels of oxytocin in the CSF, but not plasma, were significantly reduced in the peer reared animals (p = 0.019). No differences in plasma cortisol or CSF CRH were observed. Diffusion tensor imaging revealed significantly decreased white matter density across the brain. Exploratory correlational and permutation analyses suggest that the impact of peer rearing on behavior, endocrine and brain structural alterations are mediated by separate parallel mechanisms. CONCLUSIONS: Taken together, these results demonstrate in NHPs the importance of maternal rearing on the development of brain, behavior and hormonal systems that are linked to social functioning and adaptive responses. The findings suggest that the effects of maternal deprivation are mediated via multiple independent pathways which may account for the heterogeneity in behavioral and biological alterations observed in individuals that have experienced this early life adversity.

The central extended amygdala is an evolutionarily conserved set of interconnected brain regions that play an important role in threat processing to promote survival. Two core components of the central extended amygdala, the central nucleus of the amygdala (Ce) and the lateral bed nucleus of the stria terminalis (BST) are highly similar regions that serve complimentary roles by integrating fear- and anxiety-relevant information. Survival depends on the ability of the central extended amygdala to rapidly integrate and respond to threats that vary in their immediacy, proximity, and characteristics. Future studies will benefit from understanding alterations in central extended amygdala function in relation to stress-related psychopathology.

Anxiety disorders are among the most prevalent psychiatric disorders, causing significant suffering and disability. Relative to other psychiatric disorders, anxiety disorders tend to emerge early in life, supporting the importance of developmental mechanisms in their emergence and maintenance. Behavioral inhibition (BI) is a temperament that emerges early in life and, when stable and extreme, is linked to an increased risk for the later development of anxiety disorders and other stress-related psychopathology. Understanding the neural systems and molecular mechanisms underlying this dispositional risk could provide insight into treatment targets for anxiety disorders. Nonhuman primates (NHPs) have an anxiety-related temperament, called anxious temperament (AT), that is remarkably similar to BI in humans, facilitating the design of highly translational models for studying the early risk for stress-related psychopathology. Because of the recent evolutionary divergence between humans and NHPs, many of the anxiety-related brain regions that contribute to psychopathology are highly similar in terms of their structure and function, particularly with respect to the prefrontal cortex. The orbitofrontal cortex plays a critical role in the flexible encoding and regulation of threat responses, in part through connections with subcortical structures like the amygdala. Here, we explore individual differences in the transcriptional profile of cells within the region, using laser capture microdissection and single nuclear sequencing, providing insight into the molecules underlying individual differences in AT-related function of the pOFC, with a particular focus on previously implicated cellular systems, including neurotrophins and glucocorticoid signaling.

Alterations in central extended amygdala (EAc) function have been linked to anxiety, depression, and anxious temperament (AT), the early-life risk to develop these disorders. The EAc is composed of the central nucleus of the amygdala (Ce), the bed nucleus of the stria terminalis (BST), and the sublenticular extended amygdala (SLEA). Using a non-human primate model of AT and multimodal neuroimaging, the Ce and the BST were identified as key AT-related regions. Both areas are primarily comprised of GABAergic neurons and the lateral Ce (CeL) and lateral BST (BSTL) have among the highest expression of neuropeptides in the brain. Somatostatin (SST) is of particular interest because mouse studies demonstrate that SST neurons, along with corticotropin-releasing factor (CRF) neurons, contribute to a threat-relevant EAc microcircuit. Although the distribution of CeL and BSTL SST neurons has been explored in rodents, this system is not well described in non-human primates. In situ hybridization demonstrated an anterior-posterior gradient of SST mRNA in the CeL but not the BSTL of non-human primates. Triple-labeling immunofluorescence staining revealed that SST protein-expressing cell bodies are a small proportion of the total CeL and BSTL neurons and have considerable co-labeling with CRF. The SLEA exhibited strong SST mRNA and protein expression, suggesting a role for SST in mediating information transfer between the CeL and BSTL. These data provide the foundation for mechanistic non-human primate studies focused on understanding EAc function in neuropsychiatric disorders.

Anxious temperament, characterized by heightened behavioral and physiological reactivity to potential threat, is an early childhood risk factor for the later development of stress-related psychopathology. Using a well-validated nonhuman primate model, we tested the hypothesis that the prefrontal cortex (PFC) is critical in regulating the expression of primate anxiety-like behavior, as well as the function of subcortical components of the anxiety-related neural circuit. We performed aspiration lesions of a narrow strip of the posterior orbitofrontal cortex (OFC) intended to disrupt both cortex and axons entering, exiting and coursing through the pOFC, particularly those of the uncinate fasciculus (UF), a white matter tract that courses adjacent to and through this region. The OFC is of particular interest as a potential regulatory region because of its extensive reciprocal connections with amygdala, other subcortical structures and other frontal lobe regions. We validated this lesion method by demonstrating marked lesion-induced decreases in the microstructural integrity of the UF, which contains most of the fibers that connect the ventral PFC with temporal lobe structures as well as with other frontal regions. While the lesions resulted in modest decreases in threat-related behavior, they substantially decreased metabolism in components of the circuit underlying threat processing. These findings provide evidence for the importance of structural connectivity between the PFC and key subcortical structures in regulating the functions of brain regions known to be involved in the adaptive and maladaptive expression of anxiety.

Children with an extremely inhibited, anxious temperament (AT) are at increased risk for anxiety disorders and depression. Using a rhesus monkey model of early-life AT, we previously demonstrated that metabolism in the central extended amygdala (EAc), including the central nucleus of the amygdala (Ce) and bed nucleus of the stria terminalis (BST), is associated with trait-like variation in AT. Here, we use fMRI to examine relationships between Ce-BST functional connectivity and AT in a large multigenerational family pedigree of rhesus monkeys (n = 170 females and 208 males). Results demonstrate that Ce-BST functional connectivity is heritable, accounts for a significant but modest portion of the variance in AT, and is coheritable with AT. Interestingly, Ce-BST functional connectivity and AT-related BST metabolism were not correlated and accounted for non-overlapping variance in AT. Exploratory analyses suggest that Ce-BST functional connectivity is associated with metabolism in the hypothalamus and periaqueductal gray. Together, these results suggest the importance of coordinated function within the EAc for determining individual differences in AT and metabolism in brain regions associated with its behavioral and neuroendocrine components.SIGNIFICANCE STATEMENT Anxiety disorders directly impact the lives of nearly one in five people, accounting for substantial worldwide suffering and disability. Here, we use a nonhuman primate model of anxious temperament (AT) to understand the neurobiology underlying the early-life risk to develop anxiety disorders. Leveraging the same kinds of neuroimaging measures routinely used in human studies, we demonstrate that coordinated activation between the central nucleus of the amygdala and the bed nucleus of the stria terminalis is correlated with, and coinherited with, early-life AT. Understanding how these central extended amygdala regions work together to produce extreme anxiety provides a neural target for early-life interventions with the promise of preventing lifelong disability in at-risk children.

Posttraumatic stress disorder (PTSD) is a debilitating disorder that has been associated with brain abnormalities, including white matter alterations. However, little is known about the effect of treatment on these brain alterations. To investigate the course of white matter alterations in PTSD, we used a longitudinal design investigating treatment effects on white matter integrity using diffusion tensor imaging (DTI). Diffusion tensor and magnetization transfer images were obtained pre- and posttreatment from veterans with (n=39) and without PTSD (n=22). After treatment, 16 PTSD patients were remitted, and 23 had persistent PTSD based on PTSD diagnosis. The dorsal and hippocampal cingulum bundle, stria terminalis, and fornix were investigated as regions of interest. Exploratory whole-brain analyses were also performed. Groups were compared with repeated-measures ANOVA for fractional anisotropy (FA), and magnetization transfer ratio. Persistently symptomatic PTSD patients had increasing FA of the dorsal cingulum over time, and at reassessment these FA values were higher than both combat controls and the remitted PTSD group. Group-by-time interactions for FA were found in the hippocampal cingulum, fornix, and stria terminalis, posterior corona radiata, and superior longitudinal fasciculus. Our results indicate that higher FA of the dorsal cingulum bundle may be an acquired feature of persistent PTSD that develops over time. Furthermore, treatment might have differential effects on the hippocampal cingulum, fornix, stria terminalis, posterior corona radiata, and superior longitudinal fasciculus in remitted vs persistent PTSD patients. This study contributes to a better understanding of the neural underpinnings of PTSD treatment outcome.