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Background An early-life anxious temperament (AT) is a risk factor for the development of anxiety, depression, and comorbid substance abuse. We validated a nonhuman primate model of early-life AT and identified the dorsal amygdala as a core component of AT's neural circuit. Here, we combine RNA sequencing, viral-vector gene manipulation, functional brain imaging, and behavioral phenotyping to uncover AT's molecular substrates. Methods In response to potential threat, AT and brain metabolism were assessed in 46 young rhesus monkeys. We identified AT-related transcripts using RNA-sequencing data from dorsal amygdala tissue (including central nucleus of the amygdala [Ce] and dorsal regions of the basal nucleus). Based on the results, we overexpressed the neurotrophin-3 gene, NTF3, in the dorsal amygdala using intraoperative magnetic resonance imaging–guided surgery (n = 5 per group). Results This discovery-based approach identified AT-related alterations in the expression of well-established and novel genes, including an inverse association between NTRK3 expression and AT. NTRK3 is an interesting target because it is a relatively unexplored neurotrophic factor that modulates intracellular neuroplasticity pathways. Overexpression of the transcript for NTRK3's endogenous ligand, NTF3, in the dorsal amygdala resulted in reduced AT and altered function in AT's neural circuit. Conclusions Together, these data implicate neurotrophin-3/NTRK3 signaling in the dorsal amygdala in mediating primate anxiety. More generally, this approach provides an important step toward understanding the molecular underpinnings of early-life AT and will be useful in guiding the development of treatments to prevent the development of stress-related psychopathology.

BackgroundChildren exhibiting extreme anxious temperament (AT) are at an increased risk for developing anxiety and depression. Our previous mechanistic and neuroimaging work in young rhesus monkeys linked the central nucleus of the amygdala to AT and its underlying neural circuit.MethodsHere, we used laser capture microscopy and RNA sequencing in 47 young rhesus monkeys to investigate AT's molecular underpinnings by focusing on neurons from the lateral division of the central nucleus of the amygdala (CeL). RNA sequencing identified numerous AT-related CeL transcripts, and we used immunofluorescence (n= 3) and tract-tracing (n= 2) methods in a different sample of monkeys to examine the expression, distribution, and projection pattern of neurons expressing one of these transcripts.ResultsWe found 555 AT-related transcripts, 14 of which were confirmed with high statistical confidence (false discovery rate < .10), including protein kinase C delta (PKCδ), a CeL microcircuit cell marker implicated in rodent threat processing. We characterized PKCδ neurons in the rhesus CeL, compared its distribution with that of the mouse, and demonstrated that a subset of these neurons project to the laterodorsal bed nucleus of the stria terminalis.ConclusionsThese findings demonstrate that CeL PKCδ is associated with primate anxiety, provides evidence of a CeL to laterodorsal bed nucleus of the stria terminalis circuit that may be relevant to understanding human anxiety, and points to specific molecules within this circuit that could serve as potential treatment targets for anxiety disorders.

Virus-induced secretion of proinflammatory chemokines (e.g., regulated on activation, normal T cells expressed and secreted [RANTES], interleukin [IL]-8) by airway epithelial cells helps to initiate antiviral responses and airway inflammation by enhancing inflammatory cell recruitment. To define mechanisms for virus-induced chemokine secretion, monolayers of nontransformed bronchial epithelial cells were transfected or incubated with polydeoxyinosinic-deoxycytidylic acid (synthetic double-stranded [ds] RNA), rhinovirus dsRNA, or single-stranded RNA (ssRNA), and the secretion of selected chemokines was determined. Transfection or incubation with dsRNA, but not ssRNA, significantly enhanced secretion of RANTES and IL-8, but not eotaxin or macrophage inflammatory protein-1alpha. Mechanistically, dsRNA induced and activated dsRNA-dependent protein kinase (PKR), and activated nuclear factor-kappaB and p38 mitogen-activated protein kinase. Furthermore, the PKR inhibitor 2-aminopurine significantly blocked dsRNA-induced RANTES and IL-8 secretion, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 suppressed dsRNA-induced IL-8, but not RANTES. These findings indicate that dsRNA selectively induce the secretion of chemokines such as IL-8 and RANTES, and implicate dsRNA-sensitive signaling proteins in this process. Moreover, these data suggest that this may be an important mechanism for the selective secretion of chemokines by viruses (e.g., rhinovirus, respiratory syncytial virus, influenza) that synthesize dsRNA during replication.