Neuroanatomical Substrates of Circuit-Specific Cholinergic Modulation across the Primate Anterior Cingulate Cortex.

Acetylcholine is a robust neuromodulator of the limbic system and a critical regulator of arousal and emotions. The anterior cingulate cortex (ACC) and the amygdala (AMY) are key limbic structures that are both densely innervated by cholinergic afferents and interact with each other for emotional regulation. The ACC is composed of functionally distinct dorsal (A24), rostral (A32), and ventral (A25) areas that differ in their connections with the AMY. The structural substrates of cholinergic modulation of distinct ACC microcircuits and outputs to AMY are thought to depend on the laminar and subcellular localization of cholinergic receptors. The present study examines the distribution of muscarinic acetylcholine receptors, m1 and m2, on distinct excitatory and inhibitory neurons and on AMY-targeting projection neurons within ACC areas, via immunohistochemistry and injections of neural tracers into the basolateral AMY in adult rhesus monkeys of both sexes. We found that laminar densities of m1+ and m2+ expressing excitatory and inhibitory neurons depended on area and cell type. Among the ACC areas, ventral subgenual ACC A25 exhibited greater m2+ localization on presynaptic inhibitory axon terminals and greater density of m1+ and m2+ expressing AMY-targeting (tracer+) pyramidal neurons. These patterns suggest robust cholinergic disinhibition and potentiation of amygdalar outputs from the limbic ventral ACC, which may be linked to the hyperexcitability of this subgenual ACC area in depression. These findings reveal the anatomical substrate of diverse cholinergic modulation of specific ACC microcircuits and amygdalar outputs that mediate cognitive-emotional integration and dysfunctions underlying stress and affective disorders.

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, using in vitro whole-cell patch-clamp recording and intracellular filling in acute slices of ventral premotor cortex (vPMC) from rhesus monkeys (Macaca mulatta) of either sex, we demonstrate that MSC-EVs reduce injury-related physiological and morphologic changes in perilesional layer 3 pyramidal neurons. At 14-16 weeks after injury, vPMC neurons from both vehicle- and EV-treated lesioned monkeys exhibited significant hyperexcitability and predominance of inhibitory synaptic currents, compared with neurons from nonlesioned control brains. However, compared with vehicle-treated monkeys, neurons from EV-treated monkeys showed lower firing rates, greater spike frequency adaptation, and excitatory:inhibitory ratio. Further, EV treatment was associated with greater apical dendritic branching complexity, spine density, and inhibition, indicative of enhanced dendritic plasticity and filtering of signals integrated at the soma. Importantly, the degree of EV-mediated reduction of injury-related pathology in vPMC was significantly correlated with measures of behavioral recovery. These data show that EV treatment dampens injury-related hyperexcitability and restores excitatory:inhibitory balance in vPMC, thereby normalizing activity within cortical networks for motor function.SIGNIFICANCE STATEMENT Neuronal plasticity can facilitate recovery of function after cortical injury, but the underlying mechanisms and efficacy of therapeutic interventions promoting this plasticity in primates are not well understood. Our recent work has shown that intravenous infusions of mesenchymal-derived extracellular vesicles (EVs) that are involved in cell-to-cell inflammatory and trophic signaling can enhance recovery of motor function after injury in monkey primary motor cortex. This study shows that this EV-mediated enhancement of recovery is associated with amelioration of injury-related hyperexcitability and restoration of excitatory-inhibitory balance in perilesional ventral premotor cortex. These findings demonstrate the efficacy of mesenchymal EVs as a therapeutic to reduce injury-related pathologic changes in the physiology and structure of premotor pyramidal neurons and support recovery of function.

Muscarinic Acetylcholine Receptor Localization on Distinct Excitatory and Inhibitory Neurons Within the ACC and LPFC of the Rhesus Monkey.

Acetylcholine (ACh) can act on pre- and post-synaptic muscarinic receptors (mAChR) in the cortex to influence a myriad of cognitive processes. Two functionally-distinct regions of the prefrontal cortex-the lateral prefrontal cortex (LPFC) and the anterior cingulate cortex (ACC)-are differentially innervated by ascending cholinergic pathways yet, the nature and organization of prefrontal-cholinergic circuitry in primates are not well understood. Using multi-channel immunohistochemical labeling and high-resolution microscopy, we found regional and laminar differences in the subcellular localization and the densities of excitatory and inhibitory subpopulations expressing m1 and m2 muscarinic receptors, the two predominant cortical mAChR subtypes, in the supragranular layers of LPFC and ACC in rhesus monkeys (Macaca mulatta). The subset of m1+/m2+ expressing SMI-32+ pyramidal neurons labeled in layer 3 (L3) was denser in LPFC than in ACC, while m1+/m2+ SMI-32+ neurons co-expressing the calcium-binding protein, calbindin (CB) was greater in ACC. Further, we found between-area differences in laminar m1+ dendritic expression, and m2+ presynaptic localization on cortico-cortical (VGLUT1+) and sub-cortical inputs (VGLUT2+), suggesting differential cholinergic modulation of top-down vs. bottom-up inputs in the two areas. While almost all inhibitory interneurons-identified by their expression of parvalbumin (PV+), CB+, and calretinin (CR+)-expressed m1+, the localization of m2+ differed by subtype and area. The ACC exhibited a greater proportion of m2+ inhibitory neurons compared to the LPFC and had a greater density of presynaptic m2+ localized on inhibitory (VGAT+) inputs targeting proximal somatodendritic compartments and axon initial segments of L3 pyramidal neurons. These data suggest a greater capacity for m2+-mediated cholinergic suppression of inhibition in the ACC compared to the LPFC. The anatomical localization of muscarinic receptors on ACC and LPFC micro-circuits shown here contributes to our understanding of diverse cholinergic neuromodulation of functionally-distinct prefrontal areas involved in goal-directed behavior, and how these interactions maybe disrupted in neuropsychiatric and neurological conditions.

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.