DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

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.

Gene expression in the primate orbitofrontal cortex related to anxious temperament.

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.

Prefrontal influences on the function of the neural circuitry underlying anxious temperament in primates.

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.

DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

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.

The prefrontal cortex, pathological anxiety, and anxiety disorders.

Anxiety is experienced in response to threats that are distal or uncertain, involving changes in one's subjective state, autonomic responses, and behavior. Defensive and physiologic responses to threats that involve the amygdala and brainstem are conserved across species. While anxiety responses typically serve an adaptive purpose, when excessive, unregulated, and generalized, they can become maladaptive, leading to distress and avoidance of potentially threatening situations. In primates, anxiety can be regulated by the prefrontal cortex (PFC), which has expanded in evolution. This prefrontal expansion is thought to underlie primates' increased capacity to engage high-level regulatory strategies aimed at coping with and modifying the experience of anxiety. The specialized primate lateral, medial, and orbital PFC sectors are connected with association and limbic cortices, the latter of which are connected with the amygdala and brainstem autonomic structures that underlie emotional and physiological arousal. PFC pathways that interface with distinct inhibitory systems within the cortex, the amygdala, or the thalamus can regulate responses by modulating neuronal output. Within the PFC, pathways connecting cortical regions are poised to reduce noise and enhance signals for cognitive operations that regulate anxiety processing and autonomic drive. Specialized PFC pathways to the inhibitory thalamic reticular nucleus suggest a mechanism to allow passage of relevant signals from thalamus to cortex, and in the amygdala to modulate the output to autonomic structures. Disruption of specific nodes within the PFC that interface with inhibitory systems can affect the negative bias, failure to regulate autonomic arousal, and avoidance that characterize anxiety disorders.

Nonhuman Primate Models to Explore Mechanisms Underlying Early-Life Temperamental Anxiety.

Anxiety disorders are among the most prevalent psychiatric disorders, causing significant suffering and disability. Behavioral inhibition is a temperament that is linked to an increased risk for the later development of anxiety disorders and other stress-related psychopathology, and understanding the neural systems underlying this dispositional risk could provide insight into novel treatment targets for anxiety disorders. Nonhuman primates (NHPs) have anxiety-related temperaments that are similar to those of humans with behavioral inhibition, facilitating the design of translational models related to human psychopathology. Characterization of our NHP model of behavioral inhibition, which we term anxious temperament (AT), reveals that it is trait-like. Exploration of the neural substrates of AT in NHPs has revealed a distributed neural circuit that is linked to individual differences in AT, which includes the dorsal amygdala. AT-related metabolism in the dorsal amygdala, including the central nucleus, is stable across time and can be detected even in safe contexts, suggesting that AT has trait-like neural signatures within the brain. The use of lesioning and novel chemogenetic methods allows for mechanistic perturbation of the amygdala to determine its causal contribution to AT. Studies characterizing the molecular bases for individual differences in AT in the dorsal amygdala, which take advantage of novel methods for probing cellular and molecular systems, suggest involvement of neurotrophic systems, which point to the importance of neuroplasticity in AT. These novel methods, when used in combination with translational NHP models such as AT, promise to provide insights into the brain systems underlying the early risk for anxiety disorder development.