Anxious temperament (AT) is a nonhuman primate phenotype that models childhood behavioral inhibition, a prominent risk factor for anxiety disorders and stress-related psychopathology. To understand its earliest antecedents, we characterized AT's developmental trajectory over the first year of life. Infant monkeys (n = 35, 24 females) were longitudinally phenotyped for AT by assessing threat-related increases in freezing behavior, reductions in coo-calling, and increases in circulating cortisol at 1.5, 6, 12, 24, and 52 weeks. AT levels increased linearly, reaching mature levels by 52 weeks. Individual differences in AT, and its components, were stable across development. These findings support further studies during early primate life aimed to uncover neurobiological factors mediating development of this phenotype, which in humans is linked to stress-related psychopathology. SUMMARY: Anxious temperament (AT) or behavioral inhibition (BI), a trait-like characteristic in humans associated with later development of anxiety-related psychopathology, is evident during earliest weeks of primate life. Developmental patterns of AT and its components (threat-related freezing, reductions in cooing, increases in plasma cortisol) across the first year of primate life are presented. Individual differences in AT and its behavioral and hormonal components are relatively stable across the first year of life, reaching maturity by one year.

Anxiety disorders (ADs) constitute the most common psychopathology in youth, however current therapeutic options are often suboptimal and not mechanistically targeted. White matter microarchitecture, including myelination, shows promise as a potential neural correlate of pathological anxiety and modifiable treatment target, particularly in early in life when ADs commonly emerge.

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.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

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.

Myelination subserves efficient neuronal communication, and alterations in white matter (WM) microstructure have been implicated in numerous psychiatric disorders, including pathological anxiety. Recent work in rodents suggests that muscarinic antagonists may enhance myelination with behavioral benefits; however, the neural and behavioral effects of muscarinic antagonists have yet to be explored in non-human primates (NHP). Here, as a potentially translatable therapeutic strategy for human pathological anxiety, we present data from a first-in-primate study exploring the effects of the muscarinic receptor antagonist solifenacin on anxious behaviors and WM microstructure. 12 preadolescent rhesus macaques (6 vehicle control, 6 experimental; 8F, 4M) were included in a pre-test/post-test between-group study design. The experimental group received solifenacin succinate for ~60 days. Subjects underwent pre- and post-assessments of: 1) anxious temperament (AT)-related behaviors in the potentially threatening no-eye-contact (NEC) paradigm (30-min); and 2) WM and regional brain metabolism imaging metrics, including diffusion tensor imaging (DTI), quantitative relaxometry (QR), and FDG-PET. In relation to anxiety-related behaviors expressed during the NEC, significant Group (vehicle control vs. solifenacin) by Session (pre vs. post) interactions were found for freezing, cooing, and locomotion. Compared to vehicle controls, solifenacin-treated subjects exhibited effects consistent with reduced anxiety, specifically decreased freezing duration, increased locomotion duration, and increased cooing frequency. Furthermore, the Group-by-Session-by-Sex interaction indicated that these effects occurred predominantly in the males. Exploratory whole-brain voxelwise analyses of post-minus-pre differences in DTI, QR, and FDG-PET metrics revealed some solifenacin-related changes in WM microstructure and brain metabolism. These findings in NHPs support the further investigation of the utility of antimuscarinic agents in targeting WM microstructure as a means to treat pathological anxiety.

Hypothalamic-pituitary-adrenal(HPA)-activation is critical to the stress response, promoting adaptation and survival. To further understand the developmental time-course of stress-related HPA-activation and its neural correlates in nonhuman primates (NHP), we longitudinally studied infant rhesus monkeys from birth to 1-year. 35 monkeys (24 F/11 M) were assessed 5 times during development (T1-T5; average ages: 11,43,84,168,365 days). At each timepoint, subjects were injected with 18flurodeoxyglucose (FDG) and exposed to 30 minutes of potential threat (No-eye-contact condition of Human Intruder Paradigm, NEC). Subjects were anesthetized, had blood drawn, and underwent a PET scan. We performed timepoint-specific voxel-wise correlations of plasma cortisol levels and FDG-PET metabolism. Threat-induced cortisol levels displayed logarithmic growth across the first year of life (p<0.0002). Cortisol at T1 predicted cortisol levels at T2 and T3 (p<0.001), but not at T4 or T5 (p>0.1). Cortisol levels related to metabolism in different brain regions at each age (p<0.005, uncorrected). At younger ages, higher cortisol levels following NEC were associated with decreased brain metabolism (T1: amygdala; T2: amygdala, anterior hippocampus). At older ages, higher cortisol levels were associated with increased brain metabolism (T4: sublenticular extended amygdala, T5: bed nucleus of stria terminalis, thalamus). These findings demonstrate that the developmental pattern of HPA-activation in young NHPs may be logarithmic in nature. Our findings also indicate changes to the relationship between individual differences in stress-related HPA-activation and regional brain metabolism during this time. Interestingly, cortisol has negative correlations with limbic regions in newborns, later transitioning to adult-like positive correlations with limbic regions.

Cortisol is a stress hormone that mediates key physiological responses to threatening situations and modulates neural systems. However, its impact on brain structure in early-life, particularly white matter (WM) microarchitecture, remains poorly understood. In a longitudinal study of infant macaques, we use diffusion tensor imaging and quantitative relaxometry to characterize the early-life link between threat-induced cortisol and WM microarchitecture. 35 rhesus macaques were imaged with DTI and the MPnRAGE sequence on a 3T-scanner at 3, 7, 13, 25, and 53 weeks old to compute FA and qR1 (higher values~increased WM-microstructure) and MD and RD (higher values~decreased WM-microstructure). At each timepoint, subjects underwent 30 min of the no-eye-contact paradigm (NEC), in which they were exposed to an indirect threat, the profile of a human intruder. Plasma cortisol was measured after each NEC. Linear mixed-effects models assessed within-subject relations between cortisol and each WM metric in 18 WM ROIs, controlling for age-at-scan and sex. On a within-subject level (p<0.05-corrected), cortisol was positively associated with FA in 15/18 ROIs (mean-R2=0.08) and qR1 in 18/18 ROIs (mean-R2=0.12). Cortisol was negatively associated with MD in 11/18 ROIs (mean-R2=0.11) and RD in 14/18 ROIs (mean-R2=0.11). Threat-induced cortisol predicts WM microarchitecture, as measured by multimodal imaging, across the infant macaque brain. Interestingly, cortisol relates most widely to qR1 and RD, metrics thought to be relatively sensitive markers of myelin. These findings warrant further study into cortisol's role in very early WM development and neuroplasticity, particularly as it may relate to myelination processes.

Early life extreme anxiety in response to potential threat is indicative of an anxious temperament (AT) and associated with the emergence of anxiety disorders later in life. We developed a nonhuman primate (NHP) model of AT that mechanistically implicates the posterior orbitofrontal cortex (pOFC). To characterize AT-related molecular alterations in the pOFC related to AT, we performed RNA sequencing (RNASeq) on cells harvested using both laser capture microscopy (LCM) and droplet-based isolation.

Purpose In current intraoperative MRI (IMRI) methods, an iterative approach is used to aim trajectory guides at intracerebral targets: image MR‐visible features, determine current aim by fitting model to image, manipulate device, repeat. Infrequent updates are produced by such methods, compared to rapid optically tracked stereotaxy used in the operating room. Our goal was to develop a real‐time interactive IMRI method for aiming. Methods The current trajectory was computed from two points along the guide's central axis, rather than by imaging the entire device. These points were determined by correlating one‐dimensional spokes from a radial sequence with the known cross‐sectional projection of the guide. The real‐time platform RTHawk was utilized to control MR sequences and data acquisition. On‐screen updates were viewed by the operator while simultaneously manipulating the guide to align it with the planned trajectory. Accuracy was quantitated in a phantom, and in vivo validation was demonstrated in nonhuman primates undergoing preclinical gene () and cell () delivery surgeries. Results Updates were produced at 5 Hz In 10 phantom experiments at a depth of 48 mm, the cannula tip was placed with radial error of (min, mean, max) = (0.16, 0.29, 0.68) mm. Successful in vivo delivery of payloads to all 14 targets was demonstrated across nine surgeries with depths of (min, mean, max) = (33.3, 37.9, 42.5) mm. Conclusion A real‐time interactive update rate was achieved, reducing operator fatigue without compromising accuracy. Qualitative interpretation of images during aiming was rendered unnecessary by objectively computing device alignment.

ABSTRACT 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.

Alterations in white matter (WM) development are associated with many neuropsychiatric and neurodevelopmental disorders. Most MRI studies examining WM development employ diffusion tensor imaging (DTI), which relies on estimating diffusion patterns of water molecules as a reflection of WM microstructure. Quantitative relaxometry, an alternative method for characterizing WM microstructural changes, is based on molecular interactions associated with the magnetic relaxation of protons. In a longitudinal study of 34 infant non-human primates (NHP) (Macaca mulatta) across the first year of life, we implement a novel, high-resolution, T1-weighted MPnRAGE sequence to examine WM trajectories of the longitudinal relaxation rate (qR1) in relation to DTI metrics and gestational age at scan. To the best of our knowledge, this is the first study to assess developmental WM trajectories in NHPs using quantitative relaxometry and the first to directly compare DTI and relaxometry metrics during infancy. We demonstrate that qR1 exhibits robust logarithmic growth, unfolding in a posterior-anterior and medial-lateral fashion, similar to DTI metrics. On a within-subject level, DTI metrics and qR1 are highly correlated, but are largely unrelated on a between-subject level. Unlike DTI metrics, gestational age at birth (time in utero) is a strong predictor of early postnatal qR1 levels. Whereas individual differences in DTI metrics are maintained across the first year of life, this is not the case for qR1. These results point to the similarities and differences in using quantitative relaxometry and DTI in developmental studies, providing a basis for future studies to characterize the unique processes that these measures reflect at the cellular and molecular level.

White matter (WM) development early in life is a critical component of brain development that facilitates the coordinated function of neuronal pathways. Additionally, alterations in WM have been implicated in various neurodevelopmental disorders, including psychiatric disorders. Because of the need to understand WM development in the weeks immediately following birth, we characterized changes in WM microstructure throughout the postnatal macaque brain during the first year of life. This is a period in primates during which genetic, developmental, and environmental factors may have long-lasting impacts on WM microstructure. Studies in nonhuman primates (NHPs) are particularly valuable as a model for understanding human brain development because of their evolutionary relatedness to humans. Here, 34 rhesus monkeys (23 females, 11 males) were imaged longitudinally at 3, 7, 13, 25, and 53 weeks of age with T1-weighted (MPnRAGE) and diffusion tensor imaging (DTI). With linear mixed-effects (LME) modeling, we demonstrated robust logarithmic growth in FA, MD, and RD trajectories extracted from 18 WM tracts across the brain. Estimated rate of change curves for FA, MD, and RD exhibited an initial 10-week period of exceedingly rapid WM development, followed by a precipitous decline in growth rates. K-means clustering of raw DTI trajectories and rank ordering of LME model parameters revealed distinct posterior-to-anterior and medial-to-lateral gradients in WM maturation. Finally, we found that individual differences in WM microstructure assessed at 3 weeks of age were significantly related to those at 1 year of age. This study provides a quantitative characterization of very early WM growth in NHPs and lays the foundation for future work focused on the impact of alterations in early WM developmental trajectories in relation to human psychopathology.