The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. Here, we assessed over 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. While most transcriptomically-defined cell subtypes are conserved, we detected several only in some species and substantial species-specific molecular differences across homologous neuronal, glial and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin (SST) and tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine production, in certain interneurons, and also by expression of the neuropsychiatric risk gene FOXP2 , which is human-specific in microglia and primate-specific in layer-4 granular neurons. We generated a comprehensive survey of dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

The lateral division of the bed nucleus of the stria terminalis (BSTL) and central nucleus of the amygdala (Ce) form the two poles of the ‘central extended amygdala’, a theorized subcortical macrostructure important in threat-related processing. Our previous work in nonhuman primates, and humans, demonstrating strong resting fMRI connectivity between the Ce and BSTL regions, provides evidence for the integrated activity of these structures. To further understand the anatomical substrates that underlie this coordinated function, and to investigate the integrity of the central extended amygdala early in life, we examined the intrinsic connectivity between the Ce and BSTL in non-human primates using ex vivo neuronal tract tracing, and in vivo diffusion-weighted imaging and resting fMRI techniques. The tracing studies revealed that BSTL receives strong input from Ce; however, the reciprocal pathway is less robust, implying that the primate Ce is a major modulator of BSTL function. The sublenticular extended amygdala (SLEAc) is strongly and reciprocally connected to both Ce and BSTL, potentially allowing the SLEAc to modulate information flow between the two structures. Longitudinal early-life structural imaging in a separate cohort of monkeys revealed that extended amygdala white matter pathways are in place as early as 3 weeks of age. Interestingly, resting functional connectivity between Ce and BSTL regions increases in coherence from 3 to 7 weeks of age. Taken together, these findings demonstrate a time period during which information flow between Ce and BSTL undergoes postnatal developmental changes likely via direct Ce->BSTL and/or Ce <-> SLEAc <-> BSTL projections.

Background Nonhuman primate models are critical for understanding mechanisms underlying human psychopathology. We established a non-human primate model of anxious temperament (AT) for studying the early-life risk to develop anxiety and depression. Studies have identified the central nucleus of the amygdala (Ce) as an essential component of AT’s neural substrates. Corticotropin-releasing hormone (CRH) is expressed in the Ce, has a role in stress, and is linked to psychopathology. Here, in young rhesus monkeys, we combined viral vector technology with assessments of anxiety and multimodal neuroimaging to understand the consequences of chronically increased CRH in the Ce-region. Methods Using real-time intraoperative MRI-guided convection-enhanced delivery, 5 monkeys received bilateral dorsal amygdala Ce-region infusions of adeno-associated virus serotype 2 (AAV2) containing the CRH construct. Their cage-mates served as unoperated controls. AT, regional brain metabolism, “resting” fMRI, and diffusion tensor imaging (DTI) were assessed before and two months after viral infusions. Results Dorsal amygdala CRH overexpression significantly increased AT and metabolism within the dorsal amygdala. Additionally, we observed changes in metabolism in other AT-related regions, as well as in measures of functional and structural connectivity. Conclusion This study provides a translational roadmap that is important for understanding human psychopathology by combining molecular manipulations used in rodents with behavioral phenotyping and multimodal neuroimaging measures used in humans. The results indicate that chronic CRH overexpression in primates not only increases AT, but also affects metabolism and connectivity within components of AT’s neural circuitry.

Cortico-limbic circuits provide a substrate for adaptive behavioral and emotional responses. However, dysfunction of these circuits can result in maladaptive responses that are associated with psychopathology. The prefrontal-limbic pathways are of particular interest because they facilitate interactions among emotion, cognition, and decision-making functions, all of which are affected in psychiatric disorders. Regulatory aspects of the prefrontal cortex (PFC) are especially relevant to human psychopathology, as the PFC, in addition to its functions, is more recent from an evolutionary perspective and is considerably more complex in human and nonhuman primates compared with other species. This review provides a neuroanatomical and functional perspective of selected regions of the limbic system, the medial temporal lobe structures-the hippocampus and amygdala as well as regions of the PFC. Beyond the specific brain regions, emphasis is placed on the structure and function of critical PFC-limbic circuits, linking alterations in the processing of information across these pathways to the pathophysiology and psychopathology of various psychiatric illnesses.

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Contributions RK, ASF, JAO, PHR and NHK conceptualized the study. NHK and ASF oversaw the study. RK, MKR, and EMF collected behavioral data. RK and DAF developed the rapid staining laser-capture microdissection (LCM) microscopy method and collected the RNA data. DAF performed RNA extractions. JAK and his group performed RNA-Seq. TS aligned the RNA-Seq data. RK and TS analyzed the RNA-Seq data. RK, MKR, and PHR collected tissue and PHR assessed cortisol. JLF, NHK, and RK conceptualized the stereology study. RK and CEG collected and analyzed the stereology data. JLF performed retrograde tracer surgeries and collected injected tissue. RK performed triple labeling of tracing experiments and microscopic analysis. RK and NHK wrote the paper..

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 anteriorposterior 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

ContributionsRK, ASF, JAO, PHR and NHK conceptualized the study. NHK and ASF oversaw the study. RK, MKR, and EMF collected behavioral data. RK and DAF developed the rapid staining laser-capture microdissection (LCM) microscopy method and collected the RNA data. DAF performed RNA extractions. JAK and his group performed RNA-Seq. TS aligned the RNA-Seq data. RK and TS analyzed the RNA-Seq data. RK, MKR, and PHR collected tissue and PHR assessed cortisol. JLF, NHK, and RK conceptualized the stereology study. RK and CEG collected and analyzed the stereology data. JLF performed retrograde tracer surgeries and collected injected tissue. RK performed triple labeling of tracing experiments and microscopic analysis. RK and NHK wrote the paper. SummaryChildren exhibiting extreme anxious temperament (AT) are at an increased risk to develop anxiety and depression. Work in young rhesus monkeys mechanistically links the central nucleus of the amygdala (Ce) to AT. Here, we used laser capture microscopy and RNA sequencing in 47 young rhesus monkeys to investigate AT's molecular underpinnings by focusing on lateral Ce (CeL) neurons. We found 528 AT-related transcripts, including protein kinase C type-delta (PKCd), a CeL microcircuit cell marker implicated in rodent threat processing. We characterized PKCd neurons in the rhesus CeL, compared their distribution to the mouse, and demonstrated that a subset of these neurons project to the laterodorsal bed nucleus of the stria terminalis (BSTLd).These findings present evidence in the primate of a CeL to BSTLd circuit that maybe relevant to understanding human anxiety and points to specific molecules within this circuit that could serve as potential treatment targets for anxiety disorders. Keywordsfear, anxiety, stress, central nucleus of the amygdala, bed nucleus of the stria terminalis, microcircuitry, protein kinase C type delta, PKCδ, somatostatin, retrograde tracing