Cell-type specific cholinergic modulation in anterior cingulate and lateral prefrontal cortices of the rhesus macaque

The lateral prefrontal cortex (LPFC) and the anterior cingulate cortex (ACC) are two key regions of the frontal executive control network. Ascending cholinergic pathways differentially innervate these two functionally distinct cortices to modulate arousal and motivational signaling for higher-order functions. The action of acetylcholine (ACh) in sensory cortices is constrained by layer, anatomical cell type, and subcellular localization of distinct receptors, but little is known about the nature and organization of frontal-cholinergic circuitry in primates. In this dissertation, we characterized the anatomical localization of muscarinic acetylcholine receptors (mAChRs), m1 and m2–the predominant subtypes in the cortex–and their expression profiles on distinct cell types and pathways in ACC and LPFC of the rhesus monkey, using immunohistochemistry, anatomical tract-tracing, whole cell patch-clamp recordings, and single nucleus RNA sequencing. In the first series of studies (Chapter 2), we used immunohistochemistry and high-resolution confocal microscopy to reveal regional differences in m1 and m2 receptor localization on excitatory pyramidal and inhibitory neuron subpopulations and subcellular compartments in ACC (A24) versus LPFC (A46) of adult rhesus monkeys (Macaca mulatta; aged 7-11 yrs; 4 males and 2 females). The ACC exhibited a greater proportion of m2+ inhibitory neurons and a greater density of presynaptic m2+ receptors localized on inhibitory (VGAT+) terminations on pyramidal neurons compared to the LPFC. This result suggests a greater cholinergic suppression of GABAergic neurotransmission in ACC. In a second set of experiments (Chapter 3), we examined the heterogeneity of m1 and m2 laminar expression in functionally distinct ACC areas A24, A25, and A32. These differ in their connections with higher order cortical areas and limbic structures, such as the amygdala (AMY). The density of m1+ and/or m2 expressing (m1+/m2+) pyramidal neurons was significantly greater in A24 compared to A25 and to A32, while A25 exhibited a significantly greater density of m2+VGAT+ terminals. In addition, we examined the substrates for cholinergic modulation of long-range cortico-limbic processing using bidirectional neural tracers to label one specific subtype, the AMY-targeting projection neurons in these ACC areas. Compared to A24 and A32, the limbic ventral A25 had a greater density of m1+/m2+ AMY-targeting pyramidal neurons across upper layers 2-3 and deep layers 5-6, suggesting stronger cholinergic modulation of amygdalar outputs. Lastly (Chapter 4), we assessed the functional effects of cholinergic modulation on excitatory and inhibitory synaptic activity as well as the molecular signatures related to m1 and m2 receptor expression. In experiments using in vitro whole-cell patch-clamp recordings of layer 3 pyramidal neurons in ACC and LPFC, we found that application of the cholinergic agonist carbachol (CCh) significantly decreased the frequency of excitatory postsynaptic currents (EPSCs) to a greater extent in ACC A24 than in LPFC A46. Using single nucleus RNA sequencing, we found that enriched m1 and m2 transcriptional profiles in distinct cell-types and frontal areas (ACC A24 and LPFC A46) had differentially expressed genes associated with down-stream signaling cascades related to synaptic signaling and plasticity. Together, these data reveal the anatomical, functional, and transcriptomic neural substrates of diverse cholinergic modulation of local excitatory and inhibitory circuits and long-range cortico-limbic pathways in functionally-distinct ACC and LPFC frontal areas that are important for cognitive-emotional integration

Non-linear dimensionality reduction on extracellular waveforms reveals cell type diversity in premotor cortex

Cortical circuits are thought to contain a large number of cell types that coordinate to produce behavior. Current in vivo methods rely on clustering of specified features of extracellular waveforms to identify putative cell types, but these capture only a small amount of variation. Here, we develop a new method (WaveMAP) that combines non-linear dimensionality reduction with graph clustering to identify putative cell types. We apply WaveMAP to extracellular waveforms recorded from dorsal premotor cortex of macaque monkeys performing a decision-making task. Using WaveMAP, we robustly establish eight waveform clusters and show that these clusters recapitulate previously identified narrow- and broad-spiking types while revealing previously unknown diversity within these subtypes. The eight clusters exhibited distinct laminar distributions, characteristic firing rate patterns, and decision-related dynamics. Such insights were weaker when using feature-based approaches. WaveMAP therefore provides a more nuanced understanding of the dynamics of cell types in cortical circuits.https://elifesciences.org/articles/67490Published versio

Treatment with Mesenchymal-Derived Extracellular Vesicles Reduces Injury-Related Pathology in Pyramidal Neurons of Monkey Perilesional Ventral Premotor Cortex

Functional recovery after cortical injury, such as stroke, is associated with neural circuit reorganization, but the underlying mechanisms and efficacy of therapeutic interventions promoting neural plasticity in primates are not well understood. Bone marrow mesenchymal stem cell-derived extracellular vesicles (MSC-EVs), which mediate cell-to-cell inflammatory and trophic signaling, are thought be viable therapeutic targets. We recently showed, in aged female rhesus monkeys, that systemic administration of MSC-EVs enhances recovery of function after injury of the primary motor cortex, likely through enhancing plasticity in perilesional motor and premotor cortices. Here, usin