Transcriptional Profiling of Primate Central Nucleus of the Amygdala Neurons to Understand the Molecular Underpinnings of Early-Life Anxious Temperament.

Children 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. Here, 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. We 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. These 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.

Kovner R ，Souaiaia T ，Fox AS ，French DA ，Goss CE ，Roseboom PH ，Oler JA ，Riedel MK ，Fekete EM ，Fudge JL ，Knowles JA ，Kalin NH ... -  《-》

Somatostatin Gene and Protein Expression in the Non-human Primate Central Extended Amygdala.

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.

Kovner R ，Fox AS ，French DA ，Roseboom PH ，Oler JA ，Fudge JL ，Kalin NH ... -  《-》

Functional Connectivity within the Primate Extended Amygdala Is Heritable and Associated with Early-Life Anxious Temperament.

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.

Fox AS ，Oler JA ，Birn RM ，Shackman AJ ，Alexander AL ，Kalin NH ... -  《-》

Cell-type specific parallel circuits in the bed nucleus of the stria terminalis and the central nucleus of the amygdala of the mouse.

Ye J ，Veinante P 《-》

Distinct Circuits From the Central Lateral Amygdala to the Ventral Part of the Bed Nucleus of Stria Terminalis Regulate Different Fear Memory.

The ability to differentiate stimuli that predict fear is critical for survival; however, the underlying molecular and circuit mechanisms remain poorly understood. We combined transgenic mice, in vivo transsynaptic circuit-dissecting anatomical approaches, optogenetics, pharmacological methods, and electrophysiological recording to investigate the involvement of specific extended amygdala circuits in different fear memory. We identified the projections from central lateral amygdala (CeL) protein kinase C δ (PKCδ)-positive neurons and somatostatin (SST)-positive neurons to GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons in the ventral part of the bed nucleus of stria terminalis (vBNST). Prolonged optogenetic activation or inhibition of the PKCδCeL-vBNST pathway specifically reduced context fear memory, whereas the SSTCeL-vBNST pathway mainly reduced tone fear memory. Intriguingly, optogenetic manipulation of vBNST neurons that received the projection from PKCδCeL neurons exerted bidirectional regulation of context fear, whereas manipulation of vBNST neurons that received the projection from SSTCeL neurons could bidirectionally regulate both context and tone fear memory. We subsequently demonstrated the presence of δ and κ opioid receptor protein expression within the CeL-vBNST circuits, potentially accounting for the discrepancy between prolonged activation of GABAergic circuits and inhibition of downstream vBNST neurons. Finally, administration of an opioid receptor antagonist cocktail on the PKCδCeL-vBNST or SSTCeL-vBNST pathway successfully restored context or tone fear memory reduction induced by prolonged activation of the circuits. Together, these findings establish a functional role for distinct CeL-vBNST circuits in the differential regulation and appropriate maintenance of fear.

Zhu Y ，Xie SZ ，Peng AB ，Yu XD ，Li CY ，Fu JY ，Shen CJ ，Cao SX ，Zhang Y ，Chen J ，Li XM ... -  《-》