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

Abstract

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

Infant Inhibited Temperament in Primates Predicts Adult Behavior, is Heritable, and is Associated with Anxiety-Relevant Genetic Variation

“…That said, together with findings in humans, these data contribute to the rationale for further study of CTNNA2 in anxiety-related behavior in animal models, and highlight a potential molecular mechanism that may drive stable anxiety across the lifespan. More generally, our results are consistent with many molecules contributing to IT, reflecting disruption across many of the brain regions and cell-types that are thought to play a role in anxiety and threat-processing (See supplemental discussion) (31,33,34,47,48,54).…”

“…In addition to our genome-wide significant hit in CTNNA2, we relaxed the formal genome-wide significance threshold and explored variation in other genes that were marginally significantly (p<.01) associated with IT (Table S4), but do not reach genome-wide significance. Interestingly, our results revealed uncorrected associations with genetic variation in genes we've previously implicated in inhibition, including an 3' UTR variant in NTRK3 (p=0.005) and an intronic variant in PRKCD (p=.007) (33,34,54). Such associations must be considered less meaningful but can provide indications of genetic effects that deserve further study.…”

Infant inhibited temperament in primates predicts adult behavior, is heritable, and is associated with anxiety-relevant genetic variation

“…In addition to our genome-wide significant hit in CTNNA2 , we relaxed the formal genome-wide significance threshold and explored variation in other genes that were marginally significantly (p<.01) associated with IT ( Table S4 ), but do not reach genome-wide significance. Interestingly, our results revealed uncorrected associations with genetic variation in genes we've previously implicated in inhibition, including an 3' UTR variant in NTRK3 (p=0.005) and an intronic variant in PRKCD (p=.007) ( 31 , 33 , 53 ). Such associations must be considered less meaningful but can provide indications of genetic effects that deserve further study.…”

Transcriptional Profiling of Amygdala Neurons Implicates PKCδ in Primate Anxious Temperament

“…The CeL sends robust projections to the laterodorsal BST (BSTLd) and this pathway is important in threat responses. In our study, 5 we found that about half of PKCd neurons project to the BSTLd, whereas very few SST neurons project to the BSTLd. We also noted dense SST varicosities surrounding BSTLd-projecting PKCd neurons, suggesting that SST may modulate this pathway.…”

“…The CeL coordinates threat responses by integrating threatrelevant information and modulating the CeM's output though a GABAergic microcircuit comprised of neuronal subpopulations e.g., protein kinase C type delta (gene: PRKCD, protein: PKCd) and somatostatin (gene: SST, protein: SST) neurons. 4 In our recently published study, 5 we describe our efforts to understand Ce gene expression in relation to individual differences in AT. Among our top hits was PRKCD, a marker of a CeL neuronal subpopulation that is critical for threat responses.…”