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.

Mesenchymal-derived extracellular vesicles enhance microglia-mediated synapse remodeling after cortical injury in aging Rhesus monkeys.

Understanding the microglial neuro-immune interactions in the primate brain is vital to developing therapeutics for cortical injury, such as stroke or traumatic brain injury. Our previous work showed that mesenchymal-derived extracellular vesicles (MSC-EVs) enhanced motor recovery in aged rhesus monkeys following injury of primary motor cortex (M1), by promoting homeostatic ramified microglia, reducing injury-related neuronal hyperexcitability, and enhancing synaptic plasticity in perilesional cortices. A focal lesion was induced via surgical ablation of pial blood vessels over lying the cortical hand representation of M1 of aged female rhesus monkeys, that received intravenous infusions of either vehicle (veh) or EVs 24 h and again 14 days post-injury. The current study used this same cohort to address how these injury- and recovery-associated changes relate to structural and molecular interactions between microglia and neuronal synapses. Using multi-labeling immunohistochemistry, high-resolution microscopy, and gene expression analysis, we quantified co-expression of synaptic markers (VGLUTs, GLURs, VGAT, GABARs), microglia markers (Iba1, P2RY12), and C1q, a complement pathway protein for microglia-mediated synapse phagocytosis, in perilesional M1 and premotor cortices (PMC). We compared this lesion cohort to age-matched non-lesion controls (ctr). Our findings revealed a lesion-related loss of excitatory synapses in perilesional areas, which was ameliorated by EV treatment. Further, we found region-dependent effects of EVs on microglia and C1q expression. In perilesional M1, EV treatment and enhanced functional recovery were associated with increased expression of C1q + hypertrophic microglia, which are thought to have a role in debris-clearance and anti-inflammatory functions. In PMC, EV treatment was associated with decreased C1q + synaptic tagging and microglia-spine contacts. Our results suggest that EV treatment may enhance synaptic plasticity via clearance of acute damage in perilesional M1, and thereby preventing chronic inflammation and excessive synaptic loss in PMC. These mechanisms may act to preserve synaptic cortical motor networks and a balanced normative M1/PMC synaptic function to support functional recovery after injury.

Mesenchymal-Derived Extracellular Vesicles Enhance Microglia-mediated Synapse Remodeling after Cortical Injury in Rhesus Monkeys.

Understanding the microglial neuro-immune interactions in the primate brain is vital to developing therapeutics for cortical injury, such as stroke. Our previous work showed that mesenchymal-derived extracellular vesicles (MSC-EVs) enhanced motor recovery in aged rhesus monkeys post-injury of primary motor cortex (M1), by promoting homeostatic ramified microglia, reducing injury-related neuronal hyperexcitability, and enhancing synaptic plasticity in perilesional cortices. The current study addresses how these injury- and recovery-associated changes relate to structural and molecular interactions between microglia and neuronal synapses. Using multi-labeling immunohistochemistry, high resolution microscopy, and gene expression analysis, we quantified co-expression of synaptic markers (VGLUTs, GLURs, VGAT, GABARs), microglia markers (Iba-1, P2RY12), and C1q, a complement pathway protein for microglia-mediated synapse phagocytosis, in perilesional M1 and premotor cortices (PMC) of monkeys with intravenous infusions of either vehicle (veh) or EVs post-injury. We compared this lesion cohort to aged-matched non-lesion controls. Our findings revealed a lesion-related loss of excitatory synapses in perilesional areas, which was ameliorated by EV treatment. Further, we found region-dependent effects of EV on microglia and C1q expression. In perilesional M1, EV treatment and enhanced functional recovery were associated with increased expression of C1q + hypertrophic microglia, which are thought to have a role in debris-clearance and anti-inflammatory functions. In PMC, EV treatment was associated with decreased C1q + synaptic tagging and microglial-spine contacts. Our results provided evidence that EV treatment facilitated synaptic plasticity by enhancing clearance of acute damage in perilesional M1, and thereby preventing chronic inflammation and excessive synaptic loss in PMC. These mechanisms may act to preserve synaptic cortical motor networks and a balanced normative M1/PMC synaptic connectivity to support functional recovery after injury.

Neuronal properties of pyramidal cells in lateral prefrontal cortex of the aging rhesus monkey brain are associated with performance deficits on spatial working memory but not executive function.

Age-related declines in cognitive abilities occur as early as middle-age in humans and rhesus monkeys. Specifically, performance by aged individuals on tasks of executive function (EF) and working memory (WM) is characterized by greater frequency of errors, shorter memory spans, increased frequency of perseverative responses, impaired use of feedback and reduced speed of processing. However, how aging precisely differentially impacts specific aspects of these cognitive functions and the distinct brain areas mediating cognition are not well understood. The prefrontal cortex (PFC) is known to mediate EF and WM and is an area that shows a vulnerability to age-related alterations in neuronal morphology. In the current study, we show that performance on EF and WM tasks exhibited significant changes with age, and these impairments correlate with changes in biophysical properties of layer 3 (L3) pyramidal neurons in lateral LPFC (LPFC). Specifically, there was a significant age-related increase in excitability of L3 LPFC pyramidal neurons, consistent with previous studies. Further, this age-related hyperexcitability of LPFC neurons was significantly correlated with age-related decline on a task of WM, but not an EF task. The current study characterizes age-related performance on tasks of WM and EF and provides insight into the neural substrates that may underlie changes in both WM and EF with age.

Neuronal properties of pyramidal cells in lateral prefrontal cortex of the aging rhesus monkey brain are associated with performance deficits on spatial working memory but not executive function.

Age-related declines in cognitive abilities occur as early as middle-age in humans and rhesus monkeys. Specifically, performance by aged individuals on tasks of executive function (EF) and working memory (WM) is characterized by greater frequency of errors, shorter memory spans, increased frequency of perseverative responses, impaired use of feedback and reduced speed of processing. However, how aging precisely differentially impacts specific aspects of these cognitive functions and the distinct brain areas mediating cognition are not well understood. The prefrontal cortex (PFC) is known to mediate EF and WM and is an area that shows a vulnerability to age-related alterations in neuronal morphology. In the current study, we show that performance on EF and WM tasks exhibited significant changes with age and these impairments correlate with changes in biophysical properties of L3 pyramidal neurons in lateral LPFC (LPFC). Specifically, there was a significant age-related increase in excitability of Layer 3 LPFC pyramidal neurons, consistent with previous studies. Further, this age-related hyperexcitability of LPFC neurons was significantly correlated with age-related decline on a task of WM, but not an EF task. The current study characterizes age-related performance on tasks of WM and EF and provides insight into the neural substrates that may underlie changes in both WM and EF with age.

Single cell transcriptomic profiling of a neuron-astrocyte assembloid tauopathy model.

The use of iPSC derived brain organoid models to study neurodegenerative disease has been hampered by a lack of systems that accurately and expeditiously recapitulate pathogenesis in the context of neuron-glial interactions. Here we report development of a system, termed AstTau, which propagates toxic human tau oligomers in iPSC derived neuron-astrocyte assembloids. The AstTau system develops much of the neuronal and astrocytic pathology observed in tauopathies including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. Single cell transcriptomic profiling combined with immunochemistry characterizes a model system that can more closely recapitulate late-stage changes in adult neurodegeneration. The transcriptomic studies demonstrate striking changes in neuroinflammatory and heat shock protein (HSP) chaperone systems in the disease process. Treatment with the HSP90 inhibitor PU-H71 is used to address the putative dysfunctional HSP chaperone system and produces a strong reduction of pathology and neurodegeneration, highlighting the potential of AstTau as a rapid and reproducible tool for drug discovery.