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Objective: Theories of amygdala function are central to our understanding of psychiatric and neurodevelopmental disorders. However, limited knowledge of the molecular and cellular composition of the amygdala impedes translational research aimed at developing new treatments and interventions. The aim of this study was to characterize and compare the composition of amygdala cells to help bridge the gap between preclinical models and human psychiatric and neurodevelopmental disorders., Methods: Tissue was dissected from multiple amygdala subnuclei in both humans (N=3, male) and rhesus macaques (N=3, male). Single-nucleus RNA sequencing was performed to characterize the transcriptomes of individual nuclei., Results: The results reveal substantial heterogeneity between regions, even when restricted to inhibitory or excitatory neurons. Consistent with previous work, the data highlight the complexities of individual marker genes for uniquely targeting specific cell types. Cross-species analyses suggest that the rhesus monkey model is well-suited to understanding the human amygdala, but also identify limitations. For example, a cell cluster in the ventral lateral nucleus of the amygdala (vLa) is enriched in humans relative to rhesus macaques. Additionally, the data describe specific cell clusters with relative enrichment of disorder-related genes. These analyses point to the human-enriched vLa cell cluster as relevant to autism spectrum disorder, potentially highlighting a vulnerability to neurodevelopmental disorders that has emerged in recent primate evolution. Further, a cluster of cells expressing markers for intercalated cells is enriched for genes reported in human genome-wide association studies of neuroticism, anxiety disorders, and depressive disorders., Conclusions: Together, these findings shed light on the composition of the amygdala and identify specific cell types that can be prioritized in basic science research to better understand human psychopathology and guide the development of potential treatments., Competing Interests: The authors report no financial relationships with commercial interests.

A unique pool of immature glutamatergic neurons in the primate amygdala, known as the paralaminar nucleus (PL), are maturing between infancy and adolescence. The PL is a potential substrate for the steep growth curve of amygdala volume during this developmental period. A microglial component is also embedded among the PL neurons, and likely supports local neuronal maturation and emerging synaptogenesis. Microglia may alter neuronal growth following environmental perturbations such as stress. Using multiple measures, we first found that microglia in the infant primate PL had relatively large somas, and a small arbor size. In contrast, microglia in the adolescent PL had a smaller soma, and a larger dendritic arbor. We then examined microglial morphology in the PL after a novel maternal separation protocol, to examine the effects of early life stress. After maternal separation, the microglia had increased soma size, arbor size and complexity. Surprisingly, strong effects were seen not only in the infant PL, but also in the adolescent PL from subjects who had experienced the separation many years earlier. We conclude that under maternal-rearing conditions, PL microglia morphology tracks PL neuronal growth, progressing to a more 'mature' phenotype by adolescence. Maternal separation has long-lasting effects on microglia, altering their normal developmental trajectory, and resulting in a 'hyper-ramified' phenotype that persists for years. We speculate that these changes have consequences for neuronal development in young primates., Significance Statement: The paralaminar (PL) nucleus of the amygdala is an important source of plasticity, due to its unique repository of immature glutamatergic neurons. PL immature neurons mature between birth and adolescence. This process is likely supported by synaptogenesis, which requires microglia. Between infancy and adolescence in macaques, PL microglia became more dense, and shifted to a 'ramified' phenotype, consistent with increased synaptic pruning functions. Early life stress in the form of maternal separation, however, blunted this normal trajectory, leading to persistent 'parainflammatory' microglial morphologies. We speculate that early life stress may alter PL neuronal maturation and synapse formation through microglia.

Dopamine (DA) is involved in stress and stress-related illnesses, including many psychiatric disorders. Corticotropin-releasing factor (CRF) plays a role in stress responses and targets the ventral midbrain DA system, which is composed of DA and non-DA cells, and divided into specific subregions. Although CRF inputs to the midline A10 nuclei ("classic VTA") are known, in monkeys, CRF-containing terminals are also highly enriched in the expanded A10 parabrachial pigmented nucleus (PBP) and in the A8 retrorubral field subregions. We characterized CRF-labeled synaptic terminals on DA (tyrosine hydroxylase, TH+) and non-DA (TH-) cell types in the PBP and A8 regions using immunoreactive electron microscopy (EM) in male and female macaques. CRF labeling was present mostly in axon terminals, which mainly contacted TH-negative dendrites in both subregions. Most CRF-positive terminals had symmetric profiles. In both PBP and A8, CRF symmetric (putative inhibitory) synapses onto TH-negative dendrites were significantly greater than asymmetric (putative excitatory) profiles. This overall pattern was similar in males and females, despite shifts in the size of these effects between regions depending on sex. Because stress and gonadal hormone shifts can influence CRF expression, we also did hormonal assays over a 6-month time period and found little variability in basal cortisol across similarly housed animals at the same age. Together our findings suggest that at baseline, CRF-positive synaptic terminals in the primate PBP and A8 are poised to regulate DA indirectly through synaptic contacts onto non-DA neurons., (© 2023 Wiley Periodicals LLC.)

In human and nonhuman primates, the amygdala paralaminar nucleus (PL) contains immature neurons. To explore the PL's potential for cellular growth during development, we compared PL neurons in (1) infant and adolescent macaques (control, maternally-reared), and in (2) infant macaques that experienced separation from their mother in the first month of life compared to control maternally-reared infants. In maternally-reared animals, the adolescent PL had fewer immature neurons, more mature neurons, and larger immature soma volumes compared to infant PL. There were also fewer total neurons (immature plus mature) in adolescent versus infant PL, suggesting that some neurons move out of the PL by adolescence. Maternal separation did not change mean immature or mature neuron counts in infant PL. However, across all infant animals, immature neuron soma volume was strongly correlated with mature neuron counts. TBR1 mRNA, a transcript required for glutamatergic neuron maturation, is significantly reduced in the maternally-separated infant PL (DeCampo et al., 2017), and was also positively correlated with mature neuron counts in infant PL. We conclude that immature neurons gradually mature by adolescence, and that the stress of maternal separation may shift this trajectory, as revealed by correlations between TBR1 mRNA and mature neuron numbers across animals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Conflict of interest statement The authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The central extended amygdala (CEA) and ventral pallidum (VP) are involved in diverse motivated behaviors based on rodent models. These structures are conserved, but expanded, in higher primates, including human. Corticotropin releasing factor (CRF), a canonical "stress molecule" associated with the CEA and VP circuitry across species, is dynamically regulated by stress and drugs of abuse and misuse. CRF's effects on circuits critically depend on its colocation with primary "fast" transmitters, making this crucial for understanding circuit effects. We surveyed the distribution and colocalization of CRF-, VGluT2- (vesicular glutamate transporter 2), and VGAT- (vesicular GABA transporter) mRNA in specific subregions of the CEA and VP in young male monkeys. Although CRF-containing neurons were clustered in the lateral central bed nucleus (BSTLcn), the majority were broadly dispersed throughout other CEA subregions, and the VP. CRF/VGAT-only neurons were highest in the BSTLcn, lateral central amygdala nucleus (CeLcn), and medial central amygdala nucleus (CeM) (74%, 73%, and 85%, respectively). In contrast, lower percentages of CRF/VGAT only neurons populated the sublenticular extended amygdala (SLEAc), ventrolateral bed nucleus (BSTLP), and VP (53%, 54%, 17%, respectively), which had higher complements of CRF/VGAT/VGluT2-labeled neurons (33%, 29%, 67%, respectively). Thus, the majority of CRF-neurons at the "poles" (BSTLcn and CeLcn/CeM) of the CEA are inhibitory, while the "extended" BSTLP and SLEAc subregions, and neighboring VP, have a more complex profile with admixtures of "multiplexed" excitatory CRF neurons. CRF's colocalization with its various fast transmitters is likely circuit-specific, and relevant for understanding CRF actions on specific target sites. SIGNIFICANCE STATEMENT The central extended amygdala (CEA) and ventral pallidum (VP) regulate multiple motivated behaviors through differential downstream projections. The stress neuropeptide corticotropin releasing factor (CRF) is enriched in the CEA, and is thought to "set the gain" through modulatory effects on coexpressed primary transmitters. Using protein and transcript assays in monkey, we found that CRF neurons are broadly and diffusely distributed in CEA and VP. CRF mRNA + neurons colocalize with VGAT (GABA) and VGluT2 (glutamate) mRNAs in different proportions depending on subregion. CRF mRNA was also coexpressed in a subpopulation of VGAT/VGluT2 mRNA ("multiplexed") cells, which were most prominent in the VP and "pallidal"-like parts of the CEA. Heterogeneous CRF and fast transmitter coexpression across CEA/VP subregions implies circuit-specific effects., (Copyright © 2022 Fudge et al.)

The ventral midbrain is the primary source of dopamine- (DA) expressing neurons in most species. GABA-ergic and glutamatergic cell populations are intermixed among DA-expressing cells and purported to regulate both local and long-range dopamine neuron activity. Most work has been conducted in rodent models, however due to evolutionary expansion of the ventral midbrain in primates, the increased size and complexity of DA subpopulations warrants further investigation. Here, we quantified the number of DA neurons, and their GABA-ergic complement in classic DA cell groups A10 (midline ventral tegmental area nuclei [VTA] and parabrachial pigmented nucleus [PBP]), A9 (substantia nigra, pars compacta [SNc]) and A8 (retrorubral field [RRF]) in the macaque. Because the PBP is a disproportionately expanded feature of the A10 group, and has unique connectional features in monkeys, we analyzed A10 data by dividing it into 'classic' midline nuclei and the PBP. Unbiased stereology revealed total putative DA neuron counts to be 210,238 ± 17,127 (A10 = 110,319 ± 9649, A9 = 87,399 ± 7751 and A8 = 12,520 ± 827). Putative GABAergic neurons were fewer overall, and evenly dispersed across the DA subpopulations (GAD67 = 71,215 ± 5663; A10 = 16,836 ± 2743; A9 = 24,855 ± 3144 and A8 = 12,633 ± 3557). Calculating the GAD67/TH ratio for each subregion revealed differential balances of these two cell types across the DA subregions. The A8 subregion had the highest complement of GAD67-positive neurons compared to TH-positive neurons (1:1), suggesting a potentially high capacity for GABAergic inhibition of DA output in this region., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

The prefrontal cortex (PFC) and insula, amygdala, and striatum form interconnected networks that drive motivated behaviors. We previously found a connectional trend in which granularity of the ventromedial and orbital PFC/insula predicted connections to the amygdala, and also the breadth of amygdalo-striatal efferents, including projections beyond the "classic" ventral striatum. To further interrogate connectional relationships among the cortex, amygdala, and striatum, and to further define the "limbic" (amygdala-recipient) striatum, we conducted tract tracing studies in two cohorts of macaques (male n = 14, female n = 1). We focused on the cortico-amygdalo-striatal (indirect) and cortico-"limbic" striatal (direct) paths originating in the entire PFC and insula. Larger datasets and a quantitative approach revealed "cortical rules" in which cortical granularity predicts the complexity and location of projections to both the basal nucleus of the amygdala and striatum. Remarkably, projections from "cortical-like" basal nucleus to the striatum followed similar patterns. In both "direct" and "indirect" paths to the "limbic" striatum, agranular cortices formed a "foundational," broad projection, and were joined by inputs from progressively more differentiated cortices. In amygdalo-striatal paths, the ventral basal nucleus was the "foundational" input, with progressively more dorsal basal nucleus regions gradually adding inputs as the "limbic" striatum extended caudally. Together, the "indirect" and "direct" paths followed consistent principles in which cortical granularity dictated the strength and complexity of projections at their targets. Cluster analyses independently confirmed these connectional trends, and also highlighted connectional features that predicted termination in specific subregions of the basal nucleus and "limbic" striatum. SIGNIFICANCE STATEMENT The "limbic" system broadly refers to brain circuits that coordinate emotional responses. Here, we investigate circuits of the amygdala, which are involved in coding the emotional value of external cues, and their influence on the striatum. Regions of prefrontal cortex (PFC) and insula form gradients of overlapping inputs to the amygdala's basal nucleus, which feed forward to the striatum. Direct cortical inputs to these "amygdala-recipient" striatal areas are surprisingly organized according to similar principles but subtly shift from the "classic" ventral striatum to the caudal ventral striatum. Together, these distinct subsystems, cortico-amygdalo-striatal circuits and direct cortico-striatal circuits, provide substantial opportunity for different levels of internal, sensory, and external experiences to be integrated within the striatum, a major motor-behavioral interface., (Copyright © 2022 the authors.)