Biology subjects

Fudge, J. L.

Publications and source records attributed to Fudge, J. L..

10 recordsLinked to original sources

bioRxiv2026

Multiplexed dopamine neurons predominate in the ventral midbrain of young macaques

Dopamine (DA) is important in many fundamental behaviors, including positive and negative reinforcement, incentive salience, and decision-making. This behavioral diversity is now known to be due, in part, to neurotransmitter diversity, based on rodent models. To address DA neuron transmitter properties in higher species, we examined the ventral midbrain in 5 young macaques (3 male, 2 females, 3-6 years) using RNAscope in situ hybridization for tyrosine hydroxylase (TH), vesicular glutamate transporter 2 (VGluT2) and glutamic acid dehydroxylase 1 (GAD1) across the A10 (VTA; midline VTA nuclei and parabrachial nucleus; PBP), the A9 (substantia nigra, pars compact, SNc) and the A8 (retrorubral field, RRF) subregions. We followed up with immunocytochemical studies in the same cohort to infer extent of mRNA and protein matches. There were 7 mRNA phenotypes, with TH-mRNA containing cells forming the largest proportions of all neurons, as expected. Surprisingly, multiplexed TH+ neurons were much more frequent than TH-single labeled neurons overall (TH-VGluT2, 22% and TH-VGluT2-GAD1, 23% compared to TH-single labeled neurons, 19%). GAD1 mRNA co-expression mainly occurred in triple labeled cells, i.e. those with VGluT2- and TH mRNA expression. VGluT2 mRNA single-labeled neurons represented only 8%, and GAD1 mRNA single-labeled neurons comprised 20%, of the total population. Proportions of cellular phenotypes were similar across the A10-A9-A8 subregions. Most DA neurons in the young macaque contain multiple transmitters, indicating an important role for fast synaptic transmission alongside dopaminergic transmission in all subregions. We discuss the developmental and circuit implications of these findings in higher primates.

neuroscience↗

bioRxiv2026

Cortical and thalamic afferent connectomes distinguish ACC subregions of the macaque brain

In human and nonhuman primates, the anterior cingulate cortex (ACC) is an interface between the "interoceptive" and "exteroceptive" domains. The ACC contains discrete subdivisions that are distinct in cytoarchitecture and connectivity, and also serve unique functional roles. The subgenual ACC (sgACC) is a key area for arousal state modulation and processing negative emotion. Importantly, the sgACC is dysregulated in major depression and a major target for neuromodulation therapies such as deep brain stimulation. In contrast, the perigenual ACC (pgACC) is important for a host of cognitive functions, including error monitoring and social decision-making. Thus, understanding the major sources of afferent input to the sgACC and pgACC is essential for elucidating functional modulation, including in major depression and other related disorders. We took a mesoscopic connectomic approach to examine the balance of projections to sgACC and pgACC from two sources of glutamatergic input: the prefrontal cortex (PFC) and insula, and the thalamus (n=6 macaque monkeys). Using paired injections of retrograde tracers into the sgACC and pgACC and unbiased statistical clustering, we revealed that the ACC subdivisions are under the influence of vastly different cortical and thalamic drivers, i.e. heavily weighted inputs. We also found hierarchical rules (governed by cortical granularity and thalamo-cortical connectivity) by which PFC-ACC and thalamus-ACC circuits are organized. Overall, agranular PFC/insula cortices and associated thalamic nuclei are heavily weighted towards the sgACC, whereas pgACC receives a more even balance of afferents from agranular, dysgranular, and granular cortices and the thalamic nuclei with which they are associated. SIGNIFICANCE STATEMENTThe ACC contains discrete subdivisions based on cytoarchitecture and connectivity, which serve unique functional roles. The sgACC and pgACC subdivisions receive many similar inputs based on neuroimaging work. Here, we leverage higher resolution retrograde tract-tracing in macaques to examine the relative weights and relationships of multiple cortical and thalamic afferents to each region. Using unbiased analyses of labeled cells, we conclude that the balance of afferent inputs shifts from a connectome dominated by agranular cortices and their thalamic partners in sgACC, to a more balanced afferent connectome in pgACC, represented by agranular, dysgranular, and granular cortices and their broader thalamic partners. These results facilitate interpretation of functional studies, and bridge understanding of the connectional basis of psychiatric disorders.

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bioRxiv2026

Disrupted maternal care alters neural-microglia interactions in the primate paralaminar (PL) nucleus of the amygdala.

Prolonged postnatal maturation of the primate amygdala is thought to be driven, at least partially, by continued neural maturation within the paralaminar nucleus (PL). At birth, the PL is densely populated with post-mitotic glutamatergic neurons that gradually mature throughout postnatal life. This active process is likely supported by microglia, which promotes synaptic maturation. Our previous work showed that early life stress associated with maternal separation alters microglia development across the infant to adolescent transition. Here, we examined whether these morphologic microglial changes are associated with alterations in the numbers of pre-synaptic terminals (SYN1+ puncta), post-synaptic terminals (PSD95+ puncta), and putative excitatory contacts (SYN1-PSD95 colocalization), and whether these synaptic elements are engulfed by phagocytic microglia. In maternally reared animals, SYN1+ puncta, PSD95+ puncta, and putative synaptic contacts decreased, while microglial (IBA1+) volume, CD68+ content, and engulfment of synaptic elements increased, between infancy and adolescence. These findings suggest greater pruning of all synaptic elements by adolescence. Maternal separation altered this trajectory, resulting in increased phagocytic activity and engulfment of synaptic elements in infancy, but not in adolescence. Maternal separation also resulted in a 50% reduction in mature PL neurons by adolescence, suggesting maturational failure, cell loss, or both by adolescence. These findings demonstrate that early life stress disrupts normative synaptic pruning and microglia-synapse interactions in the developing primate PL. Increased synaptic engulfment in infants with disrupted care is associated with premature, aberrant pruning, and highlights a potential cellular mechanism through which early environmental insults could change PL neural development by adolescence. Significance StatementThe paralaminar nucleus (PL) of the amygdala is an important substrate for the delayed post-natal development of the amygdala in human and nonhuman primates. Gradually maturing glutamatergic neurons in this region, and the microglia that support them, are exposed to life events which may shape their development. We recently found that maternal separation in infants produces aberrant hyper-ramified microglia in the PL beginning in infancy and persisting into adolescence. Examining neuron-microglial interactions in the same cohort, we now find Increased phagocytic engulfment of synaptic elements by microglia after maternal separation in infancy only, with a reduction in PL mature neurons that is apparent by adolescence. Together these data suggest a mechanism for altered PL maturation, instigated by disrupted maternal care.

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bioRxiv2024

Microglia morphology in the developing primate amygdala and effects of early life stress

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 StatementThe 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.

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bioRxiv2023

Immature neurons in the primate amygdala: changes with early development and disrupted early environment

In human and nonhuman primates, the amygdala paralaminar nucleus (PL) contains immature neurons. To explore the PLs potential for cellular growth during development, we compared PL cells 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. 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. tbr-1 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 tbr1mRNA and mature neuron numbers across animals.

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bioRxiv2022

ULTRASTRUCTURAL ANALYSIS OF CRF-DA INTERACTIONS IN MALE AND FEMALE MACAQUE: BEYOND THE CLASSIC VTA

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 immuno-reactive 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 inhibitory (symmetric) profiles. In both PBP and A8, CRF symmetric (inhibitory) synapses onto TH-negative dendrites were significantly greater than asymmetric (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.

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bioRxiv2022

Unbiased stereologic estimates of dopaminergic and GABAergic neurons in the A10, A9, and A8 subpopulations in the young male Macaque

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 +/- 9,649, A9= 87,399 +/-7,751 and A8=12,520 +/- 827). Putative GABAergic neurons were fewer overall, and evenly dispersed across the DA subpopulations (GAD67= 71,215 +/- 5,663; A10=16,836 +/- 2,743; A9=24,855 +/- 3,144 and A8=12,633 +/- 3,557). 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. HIGHLIGHTSO_LIThe A10 subregion expands laterally and caudally in nonhuman primates C_LIO_LIThe A10, A9, and A8 comprise 52%, 42% and 6% of DA neurons, respectively C_LIO_LIGABAergic neurons are more evenly dispersed across subregions C_LIO_LIThe A8 subpopulation has the highest ratio of GABA: DA neurons C_LI

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bioRxiv2021

Perigenual and Subgenual Anterior Cingulate Afferents Converge on Common Pyramidal Cells in Amygdala Subregions

The subgenual (sgACC) and pregenual (pgACC) anterior cingulate are important afferents of the amygdala, with different cytoarchitecture, connectivity, and function. The sgACC is associated with arousal mechanisms linked to salient cues, while the pgACC is engaged in conflict decision-making, including in social contexts. After placing same-size, small volume tracer injections into sgACC and pgACC of the same hemisphere in male Macaques, we examined anterogradely labeled fiber distribution to understand how these different functional systems communicate in the main amygdala nuclei at both mesocopic and cellular levels. The sgACC has broad-based termination patterns. In contrast, the pgACC has a more restricted pattern which was always nested in sgACC terminals. Terminal overlap occurred in subregions of the accessory basal and basal nuclei, which we termed hotspots. In triple-labeling confocal studies, the majority of randomly selected CAMKII (+) cells (putative amygdala glutamatergic neurons) in hotspots received dual contacts from the sgACC and pgACC. The ratio of dual contacts occurred over a surprisingly narrow range, suggesting a consistent, tight balance of afferent contacts on postsynaptic neurons. Large boutons, which are associated with greater synaptic strength, were approximately 3 times more frequent on sgACC versus pgACC axon terminals in hotspots, consistent with a fast driver function. Together, the results reveal a nested interaction in which pgACC (conflict/social monitoring) terminals converge with the broader sgACC (salience) terminals at both the mesoscopic and cellular level. The pre-synaptic organization in hotspots suggest that shifts in arousal states can rapidly, and flexibly influence decision-making functions in the amygdala. Significance statementThe subgenual (sgACC) and perigenual cingulate (pgACC) have distinct structural and functional characteristics and are important afferent modulators of the amygdala. The sgACC is critical for arousal, while the pgACC mediates conflict-monitoring, including in social contexts. Using dual tracer injections in the same monkey, we found that sgACC inputs broadly project in the main amygdala nuclei, whereas pgACC inputs were more restricted and nested in zones containing sgACC terminals ( hotspots). The majority of CAMKII + (excitatory) amygdala neurons in hotspots received converging contacts, which were tightly balanced. pgACC and sgACC afferent streams are therefore highly interdependent in these specific amygdala subregions, permitting internal arousal states to rapidly shape responses of amygdala neurons involved in conflict and social monitoring networks.

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bioRxiv2021

Cortical granularity shapes information flow to the amygdala and its striatal targets in nonhuman primate

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 data sets 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 StatementThe 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 and insula form gradients of overlapping inputs to the amygdalas 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-amygdala-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.

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bioRxiv2019

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 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 (PKC{delta}), a CeL microcircuit cell marker implicated in rodent threat processing. We characterized PKC{delta} 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.

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