Key Roles of CACNA1C/Cav1.2 and CALB1/Calbindin in Prefrontal Neurons Altered in Cognitive Disorders.

Importance: The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. Objective: To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. Design, Setting, and Participants: The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Main Outcomes and Measures: Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Results: Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by ß1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or ß1-adrenoceptor antagonist protected working memory from stress. Conclusions and Relevance: The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.

The meso-connectomes of mouse, marmoset, and macaque: network organization and the emergence of higher cognition.

The recent publications of the inter-areal connectomes for mouse, marmoset, and macaque cortex have allowed deeper comparisons across rodent vs. primate cortical organization. In general, these show that the mouse has very widespread, "all-to-all" inter-areal connectivity (i.e. a "highly dense" connectome in a graph theoretical framework), while primates have a more modular organization. In this review, we highlight the relevance of these differences to function, including the example of primary visual cortex (V1) which, in the mouse, is interconnected with all other areas, therefore including other primary sensory and frontal areas. We argue that this dense inter-areal connectivity benefits multimodal associations, at the cost of reduced functional segregation. Conversely, primates have expanded cortices with a modular connectivity structure, where V1 is almost exclusively interconnected with other visual cortices, themselves organized in relatively segregated streams, and hierarchically higher cortical areas such as prefrontal cortex provide top-down regulation for specifying precise information for working memory storage and manipulation. Increased complexity in cytoarchitecture, connectivity, dendritic spine density, and receptor expression additionally reveal a sharper hierarchical organization in primate cortex. Together, we argue that these primate specializations permit separable deconstruction and selective reconstruction of representations, which is essential to higher cognition.

An overview of preclinical models of traumatic brain injury (TBI): relevance to pathophysiological mechanisms.

Background: Traumatic brain injury (TBI) is a major cause of morbidity and mortality, affecting millions annually worldwide. Although the majority of TBI patients return to premorbid baseline, a subset of patient can develop persistent and often debilitating neurocognitive and behavioral changes. The etiology of TBI within the clinical setting is inherently heterogenous, ranging from sport related injuries, fall related injuries and motor vehicle accidents in the civilian setting, to blast injuries in the military setting. Objective: Animal models of TBI, offer the distinct advantage of controlling for injury modality, duration and severity. Furthermore, preclinical models of TBI have provided the necessary temporal opportunity to study the chronic neuropathological sequelae of TBI, including neurodegenerative sequelae such as tauopathy and neuroinflammation within the finite experimental timeline. Despite the high prevalence of TBI, there are currently no disease modifying regimen for TBI, and the current clinical treatments remain largely symptom based. The preclinical models have provided the necessary biological substrate to examine the disease modifying effect of various pharmacological agents and have imperative translational value. Methods: The current review will include a comprehensive survey of well-established preclinical models, including classic preclinical models including weight drop, blast injury, fluid percussion injury, controlled cortical impact injury, as well as more novel injury models including closed-head impact model of engineered rotational acceleration (CHIMERA) models and closed-head projectile concussive impact model (PCI). In addition to rodent preclinical models, the review will include an overview of other species including large animal models and Drosophila. Results: There are major neuropathological perturbations post TBI captured in various preclinical models, which include neuroinflammation, calcium dysregulation, tauopathy, mitochondrial dysfunction and oxidative stress, axonopathy, as well as glymphatic system disruption. Conclusion: The preclinical models of TBI continue to offer valuable translational insight, as well as essential neurobiological basis to examine specific disease modifying therapeutic regimen.

Nanoscale imaging of pT217-tau in aged rhesus macaque entorhinal and dorsolateral prefrontal cortex: Evidence of interneuronal trafficking and early-stage neurodegeneration.

INTRODUCTION: Tau phosphorylated at threonine-217 (pT217-tau) is a novel fluid-based biomarker that predicts onset of Alzheimer's disease (AD) symptoms, but little is known about how pT217-tau arises in the brain, as soluble pT217-tau is dephosphorylated post mortem in humans. METHODS: We used multilabel immunofluorescence and immunoelectron microscopy to examine the subcellular localization of early-stage pT217-tau in entorhinal and prefrontal cortices of aged macaques with naturally occurring tau pathology and assayed pT217-tau levels in plasma. RESULTS: pT217-tau was aggregated on microtubules within dendrites exhibiting early signs of degeneration, including autophagic vacuoles. It was also seen trafficking between excitatory neurons within synapses on spines, where it was exposed to the extracellular space, and thus accessible to cerebrospinal fluid (CSF)/blood. Plasma pT217-tau levels increased across the age span and thus can serve as a biomarker in macaques. DISCUSSION: These data help to explain why pT217-tau predicts degeneration in AD and how it gains access to CSF and plasma to serve as a fluid biomarker.

Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability in Working Memory Circuits.

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+ influx through HCN channels activates Slack Na+-activated K+ (KNa) channels to hyperpolarize the membrane. We have found that HCN and Slack KNa channels co-immunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNa current in pyramidal cells that express both HCN and Slack channels, but has no effect on KNa currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K+ current indirectly by lowering Na+ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca2+ reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.

Nanoscale imaging of pT217-tau in aged rhesus macaque entorhinal and dorsolateral prefrontal cortex: Evidence of interneuronal trafficking and early-stage neurodegeneration.

INTRODUCTION: pT217-tau is a novel fluid-based biomarker that predicts onset of Alzheimer's disease (AD) symptoms, but little is known about how pT217-tau arises in brain, as soluble pT217-tau is dephosphorylated postmortem in humans. METHODS: We utilized multi-label immunofluorescence and immunoelectron-microscopy to examine the subcellular localization of early-stage pT217-tau in entorhinal and prefrontal cortices of aged macaques with naturally-occurring tau pathology and assayed pT217-tau levels in plasma. RESULTS: pT217-tau was aggregated on microtubules within dendrites exhibiting early signs of degeneration, including autophagic vacuoles. It was also seen trafficking between excitatory neurons within synapses on spines, where it was exposed to the extracellular space, and thus accessible to CSF/blood. Plasma pT217-tau levels increased across the age-span and thus can serve as a biomarker in macaques. DISCUSSION: These data help to explain why pT217-tau predicts degeneration in AD and how it gains access to CSF and plasma to serve as a fluid biomarker.

Chronic GCPII (glutamate-carboxypeptidase-II) inhibition reduces pT217Tau levels in the entorhinal and dorsolateral prefrontal cortices of aged macaques.

Introduction: Current approaches for treating sporadic Alzheimer's disease (sAD) focus on removal of amyloid beta 1-42 (Aß1-42) or phosphorylated tau, but additional strategies are needed to reduce neuropathology at earlier stages prior to neuronal damage. Longstanding data show that calcium dysregulation is a key etiological factor in sAD, and the cortical neurons most vulnerable to tau pathology show magnified calcium signaling, for example in dorsolateral prefrontal cortex (dlPFC) and entorhinal cortex (ERC). In primate dlPFC and ERC, type 3 metabotropic glutamate receptors (mGluR3s) are predominately post-synaptic, on spines, where they regulate cAMP-calcium signaling, a process eroded by inflammatory glutamate carboxypeptidase II (GCPII) actions. The current study tested whether enhancing mGluR3 regulation of calcium via chronic inhibition of GCPII would reduce tau hyperphosphorylation in aged macaques with naturally-occurring tau pathology. Methods: Aged rhesus macaques were treated daily with the GCPII inhibitor, 2-MPPA (2-3-mercaptopropyl-penanedioic acid (2-MPPA)),Aged rhesus macaques were treated daily with the GCPII inhibitor, 2-MPPA (2-3-mercaptopropyl-penanedioic acid (2-MPPA)). Results: Aged macaques that received 2-MPPA had significantly lower pT217Tau levels in dlPFC and ERC, and had lowered plasma pT217Tau levels from baseline. pT217Tau levels correlated significantly with GCPII activity in dlPFC. Both 2-MPPA- and vehicle-treated monkeys showed cognitive improvement; 2-MPPA had no apparent side effects. Exploratory CSF analyses indicated reduced pS202Tau with 2-MPPA administration, confirmed in dlPFC samples. Discussion: These data provide proof-of-concept support that GCPII inhibition can reduce tau hyperphosphorylation in the primate cortices most vulnerable in sAD. GCPII inhibition may be particularly helpful in reducing the risk of sAD caused by inflammation. These data in nonhuman primates should encourage future research on this promising mechanism. Highlights: Inflammation is a key driver of sporadic Alzheimer's disease.GCPII inflammatory signaling in brain decreases mGluR3 regulation of calcium.Chronic inhibition of GCPII inflammatory signaling reduced pT217Tau in aged monkeys.GCPII inhibition is a novel strategy to help prevent tau pathology at early stages.

Localization of PDE4D, HCN1 channels, and mGluR3 in rhesus macaque entorhinal cortex may confer vulnerability in Alzheimer's disease.

Alzheimer's disease cortical tau pathology initiates in the layer II cell clusters of entorhinal cortex, but it is not known why these specific neurons are so vulnerable. Aging macaques exhibit the same qualitative pattern of tau pathology as humans, including initial pathology in layer II entorhinal cortex clusters, and thus can inform etiological factors driving selective vulnerability. Macaque data have already shown that susceptible neurons in dorsolateral prefrontal cortex express a "signature of flexibility" near glutamate synapses on spines, where cAMP-PKA magnification of calcium signaling opens nearby potassium and hyperpolarization-activated cyclic nucleotide-gated channels to dynamically alter synapse strength. This process is regulated by PDE4A/D, mGluR3, and calbindin, to prevent toxic calcium actions; regulatory actions that are lost with age/inflammation, leading to tau phosphorylation. The current study examined whether a similar "signature of flexibility" expresses in layer II entorhinal cortex, investigating the localization of PDE4D, mGluR3, and HCN1 channels. Results showed a similar pattern to dorsolateral prefrontal cortex, with PDE4D and mGluR3 positioned to regulate internal calcium release near glutamate synapses, and HCN1 channels concentrated on spines. As layer II entorhinal cortex stellate cells do not express calbindin, even when young, they may be particularly vulnerable to magnified calcium actions and ensuing tau pathology.

Interrogating the Etiology of Sporadic Alzheimer's Disease Using Aging Rhesus Macaques: Cellular, Molecular, and Cortical Circuitry Perspectives.

Aging is the most significant risk factor for neurodegenerative disorders such as Alzheimer's disease (AD) associated with profound socioeconomic and personal costs. Consequently, there is an urgent need for animal models that recapitulate the age-related spatial and temporal complexity and patterns of pathology identical to human AD. Our research in aging nonhuman primate models involving rhesus macaques has revealed naturally occurring amyloid and tau pathology, including the formation of amyloid plaques and neurofibrillary tangles comprising hyperphosphorylated tau. Moreover, rhesus macaques exhibit synaptic dysfunction in association cortices and cognitive impairments with advancing age, and thus can be used to interrogate the etiological mechanisms that generate neuropathological cascades in sporadic AD. Particularly, unique molecular mechanisms (eg, feedforward cyclic adenosine 3',5'-monophosphate [cAMP]-Protein kinase A (PKA)-calcium signaling) in the newly evolved primate dorsolateral prefrontal cortex are critical for persistent firing required for subserving higher-order cognition. For example, dendritic spines in primate dorsolateral prefrontal cortex contain a specialized repertoire of proteins to magnify feedforward cAMP-PKA-calcium signaling such as N-methyl-d-aspartic acid receptors and calcium channels on the smooth endoplasmic reticulum (eg, ryanodine receptors). This process is constrained by phosphodiesterases (eg, PDE4) that hydrolyze cAMP and calcium-buffering proteins (eg, calbindin) in the cytosol. However, genetic predispositions and age-related insults exacerbate feedforward cAMP-Protein kinase A-calcium signaling pathways that induce a myriad of downstream effects, including the opening of K+ channels to weaken network connectivity, calcium-mediated dysregulation of mitochondria, and activation of inflammatory cascades to eliminate synapses, thereby increasing susceptibility to atrophy. Therefore, aging rhesus macaques provide an invaluable model to explore novel therapeutic strategies in sporadic AD.

Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability and Working Memory.

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+ influx through HCN channels activates Slack Na+-activated K+ (KNa) channels to hyperpolarize the membrane. We have found that HCN and Slack KNa channels coimmunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNa current in pyramidal cells that express both HCN and Slack channels, but has no effect on KNa currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K+ +current indirectly by lowering Na+ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca2+ reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability.

Inhibition of glutamate-carboxypeptidase-II in dorsolateral prefrontal cortex: potential therapeutic target for neuroinflammatory cognitive disorders.

Glutamate carboxypeptidase-II (GCPII) expression in brain is increased by inflammation, e.g. by COVID19 infection, where it reduces NAAG stimulation of metabotropic glutamate receptor type 3 (mGluR3). GCPII-mGluR3 signaling is increasingly linked to higher cognition, as genetic alterations that weaken mGluR3 or increase GCPII signaling are associated with impaired cognition in humans. Recent evidence from macaque dorsolateral prefrontal cortex (dlPFC) shows that mGluR3 are expressed on dendritic spines, where they regulate cAMP-PKA opening of potassium (K+) channels to enhance neuronal firing during working memory. However, little is known about GCPII expression and function in the primate dlPFC, despite its relevance to inflammatory disorders. The present study used multiple label immunofluorescence and immunoelectron microscopy to localize GCPII in aging macaque dlPFC, and examined the effects of GCPII inhibition on dlPFC neuronal physiology and working memory function. GCPII was observed in astrocytes as expected, but also on neurons, including extensive expression in dendritic spines. Recordings in dlPFC from aged monkeys performing a working memory task found that iontophoresis of the GCPII inhibitors 2-MPPA or 2-PMPA markedly increased working memory-related neuronal firing and spatial tuning, enhancing neural representations. These beneficial effects were reversed by an mGluR2/3 antagonist, or by a cAMP-PKA activator, consistent with mGluR3 inhibition of cAMP-PKA-K+ channel signaling. Systemic administration of the brain penetrant inhibitor, 2-MPPA, significantly improved working memory performance without apparent side effects, with largest effects in the oldest monkeys. Taken together, these data endorse GCPII inhibition as a potential strategy for treating cognitive disorders associated with aging and/or neuroinflammation.

Glutamate Metabotropic Receptor Type 3 (mGlu3) Localization in the Rat Prelimbic Medial Prefrontal Cortex.

Metabotropic glutamate receptors type 3 (mGlu3, encoded by GRM3) are increasingly related to cognitive functioning, including the working memory operations of the prefrontal cortex (PFC). In rhesus monkeys, mGlu3 are most commonly expressed on glia (36%), but are also very prominent on layer III dendritic spines (23%) in the dorsolateral PFC (dlPFC) where they enhance working memory-related neuronal firing. In contrast, mGlu2 are predominately presynaptic in layer III of macaque dlPFC, indicating a pre- vs. post-synaptic dissociation by receptor subtype. The current study examined the cellular and subcellular localizations of mGlu3 in the rat prelimbic medial PFC (PL mPFC), a region needed for spatial working memory performance in rodents. Multiple label immunofluorescence demonstrated mGlu3 expression in neurons and astrocytes, with rare labeling in microglia. Immunoelectron microscopy of layers III and V found that the predominant location for mGlu3 was on axons (layer III: 35.9%; layer V: 44.1%), with labeling especially prominent within the intervaricose segments distant from axon terminals. mGlu3 were also found on glia (likely astrocytes), throughout the glial membrane (layer III: 28.2%; layer V: 29.5%). Importantly, mGlu3 could be seen on dendritic spines, especially in layer III (layer III: 15.6%; layer V: 8.2%), with minor labeling on dendrites. These data show that there are some similarities between mGlu3 expression in rat PL mPFC and macaque dlPFC, but the spine expression enriches and differentiates in the more recently evolved primate dlPFC.

Unusual Molecular Regulation of Dorsolateral Prefrontal Cortex Layer III Synapses Increases Vulnerability to Genetic and Environmental Insults in Schizophrenia.

Schizophrenia is associated with reduced numbers of spines and dendrites from layer III of the dorsolateral prefrontal cortex (dlPFC), the layer that houses the recurrent excitatory microcircuits that subserve working memory and abstract thought. Why are these synapses so vulnerable, while synapses in deeper or more superficial layers are little affected? This review describes the special molecular properties that govern layer III neurotransmission and neuromodulation in the primate dlPFC and how they may render these circuits particularly vulnerable to genetic and environmental insults. These properties include a reliance on NMDA receptor rather than AMPA receptor neurotransmission; cAMP (cyclic adenosine monophosphate) magnification of calcium signaling near the glutamatergic synapse of dendritic spines; and potassium channels opened by cAMP/PKA (protein kinase A) signaling that dynamically alter network strength, with built-in mechanisms to take dlPFC "offline" during stress. A variety of genetic and/or environmental insults can lead to the same phenotype of weakened layer III connectivity, in which mechanisms that normally strengthen connectivity are impaired and those that normally weaken connectivity are intensified. Inflammatory mechanisms, such as increased kynurenic acid and glutamate carboxypeptidase II expression, are especially detrimental to layer III dlPFC neurotransmission and modulation, mimicking genetic insults. The combination of genetic and inflammatory insults may cross the threshold into pathology.

Glutamate Carboxypeptidase II in Aging Rat Prefrontal Cortex Impairs Working Memory Performance.

Glutamate carboxypeptidase II (GCPII) expression in brain is increased by inflammation, and reduces NAAG (N-acetyl aspartyl glutamate) stimulation of mGluR3 signaling. Genetic insults in this signaling cascade are increasingly linked to cognitive disorders in humans, where increased GCPII and or decreased NAAG-mGluR3 are associated with impaired prefrontal cortical (PFC) activation and cognitive impairment. As aging is associated with increased inflammation and PFC cognitive deficits, the current study examined GCPII and mGluR3 expression in the aging rat medial PFC, and tested whether GCPII inhibition with 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA) would improve working memory performance. We found that GCPII protein was expressed on astrocytes and some microglia as expected from previous studies, but was also prominently expressed on neurons, and showed increased levels with advancing age. Systemic administration of the GCPII inhibitor, 2-MPPA, improved working memory performance in young and aged rats, and also improved performance after local infusion into the medial PFC. As GCPII inhibitors are well-tolerated, they may provide an important new direction for treatment of cognitive disorders associated with aging and/or inflammation.

Simple, Single-Shot Phosphoproteomic Analysis of Heat-Stable Tau Identifies Age-Related Changes in pS235- and pS396-Tau Levels in Non-human Primates.

Age is the most significant risk factor for Alzheimer's disease (AD), and understanding its role in specific aspects of AD pathology will be critical for therapeutic development. Neurofibrillary tangles composed of hyperphosphorylated tau are a quintessential hallmark of AD. To study age-related changes in tau phosphorylation, we developed a simple, antibody-free approach for single shot analysis of tau phosphorylation across the entire protein by liquid-chromatography tandem mass spectrometry. This methodology is species independent; thus, while initially developed in a rodent model, we utilized this technique to analyze 36 phosphorylation sites on rhesus monkey tau from the prefrontal cortex (PFC), a region vulnerable to AD-linked degeneration. Data are available via ProteomeXchange with identifier PXD027971. We identified novel, age-related changes in tau phosphorylation in the rhesus monkey PFC and analyzed patterns of phosphorylation change across domains of the protein. We confirmed a significant increase and positive correlation with age of phosphorylated serine 235 tau and phosphorylated serine 396 tau levels in an expanded cohort of 14 monkeys. Histology showed robust labeling for tau phosphorylated at these sites in vulnerable layer III pyramidal cells in the PFC. The results presented in this study suggest an important role of the natural aging process in tau phosphorylation in rhesus monkey.

Chronic Stress Weakens Connectivity in the Prefrontal Cortex: Architectural and Molecular Changes.

Chronic exposure to uncontrollable stress causes loss of spines and dendrites in the prefrontal cortex (PFC), a recently evolved brain region that provides top-down regulation of thought, action, and emotion. PFC neurons generate top-down goals through recurrent excitatory connections on spines. This persistent firing is the foundation for higher cognition, including working memory, and abstract thought. However, exposure to acute uncontrollable stress drives high levels of catecholamine release in the PFC, which activates feedforward calcium-cAMP signaling pathways to open nearby potassium channels, rapidly weakening synaptic connectivity to reduce persistent firing. Chronic stress exposures can further exacerbate these signaling events leading to loss of spines and resulting in marked cognitive impairment. In this review, we discuss how stress signaling mechanisms can lead to spine loss, including changes to BDNF-mTORC1 signaling, calcium homeostasis, actin dynamics, and mitochondrial actions that engage glial removal of spines through inflammatory signaling. Stress signaling events may be amplified in PFC spines due to cAMP magnification of internal calcium release. As PFC dendritic spine loss is a feature of many cognitive disorders, understanding how stress affects the structure and function of the PFC will help to inform strategies for treatment and prevention.

Studies of aging nonhuman primates illuminate the etiology of early-stage Alzheimer's-like neuropathology: An evolutionary perspective.

Tau pathology in Alzheimer's disease (AD) preferentially afflicts the limbic and recently enlarged association cortices, causing a progression of mnemonic and cognitive deficits. Although genetic mouse models have helped reveal mechanisms underlying the rare, autosomal-dominant forms of AD, the etiology of the more common, sporadic form of AD remains unknown, and is challenging to study in mice due to their limited association cortex and lifespan. It is also difficult to study in human brains, as early-stage tau phosphorylation can degrade postmortem. In contrast, rhesus monkeys have extensive association cortices, are long-lived, and can undergo perfusion fixation to capture early-stage tau phosphorylation in situ. Most importantly, rhesus monkeys naturally develop amyloid plaques, neurofibrillary tangles comprised of hyperphosphorylated tau, synaptic loss, and cognitive deficits with advancing age, and thus can be used to identify the early molecular events that initiate and propel neuropathology in the aging association cortices. Studies to date suggest that the particular molecular signaling events needed for higher cognition-for example, high levels of calcium to maintain persistent neuronal firing- lead to tau phosphorylation and inflammation when dysregulated with advancing age. The expression of NMDAR-NR2B (GluN2B)-the subunit that fluxes high levels of calcium-increases over the cortical hierarchy and with the expansion of association cortex in primate evolution, consistent with patterns of tau pathology. In the rhesus monkey dorsolateral prefrontal cortex, spines contain NMDAR-NR2B and the molecular machinery to magnify internal calcium release near the synapse, as well as phosphodiesterases, mGluR3, and calbindin to regulate calcium signaling. Loss of regulation with inflammation and/or aging appears to be a key factor in initiating tau pathology. The vast expansion in the numbers of these synapses over primate evolution is consistent with the degree of tau pathology seen across species: marmoset < rhesus monkey < chimpanzee < human, culminating in the vast neurodegeneration seen in humans with AD.

Age-related calcium dysregulation linked with tau pathology and impaired cognition in non-human primates.

INTRODUCTION: The etiology of sporadic Alzheimer's disease (AD) requires non-genetically modified animal models. METHODS: The relationship of tau phosphorylation to calcium-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) dysregulation was analyzed in aging rhesus macaque dorsolateral prefrontal cortex (dlPFC) and rat primary cortical neurons using biochemistry and immuno-electron microscopy. The influence of calcium leak from ryanodine receptors (RyRs) on neuronal firing and cognitive performance was examined in aged macaques. RESULTS: Aged monkeys naturally develop hyperphosphorylated tau, including AD biomarkers (AT8 (pS202/pT205) and pT217) and early tau pathology markers (pS214 and pS356) that correlated with evidence of increased calcium leak (pS2808-RyR2). Calcium also regulated early tau phosphorylation in vitro. Age-related reductions in the calcium-binding protein, calbindin, and in phosphodiesterase PDE4D were seen within dlPFC pyramidal cell dendrites. Blocking RyRs with S107 improved neuronal firing and cognitive performance in aged macaques. DISCUSSION: Dysregulated calcium signaling confers risk for tau pathology and provides a potential therapeutic target.

M1 receptors interacting with NMDAR enhance delay-related neuronal firing and improve working memory performance.

The recurrent excitatory circuits in dlPFC underlying working memory are known to require activation of glutamatergic NMDA receptors (NMDAR). The neurons in these circuits also rely on acetylcholine to maintain persistent activity, with evidence for actions at both nicotinic α7 receptors and muscarinic M1 receptors (M1R). It is known that nicotinic α7 receptors interact with NMDAR in these circuits, but the interactions between M1R and NMDAR on dlPFC neuronal activity are unknown. Here, we investigated whether M1Rs contribute to the permissive effects of ACh in dlPFC circuitry underlying working memory via interactions with NMDA receptors. We tested interactions between M1Rs and NMDARs in vivo on single neuron activity in rhesus macaques performing a working memory task, as well as on working memory behavior in rodents following infusion of M1R and NMDAR compounds into mPFC. We report that M1R antagonists block the enhancing effects of NMDA application, consistent with M1R permissive actions. Conversely, M1R positive allosteric modulators prevented the detrimental effects of NMDAR blockade in single neurons in dlPFC and on working memory performance in rodents. These data support an interaction between M1R and NMDARs in working memory circuitry in both primates and rats, and suggest M1Rs contribute to the permissive actions of ACh in primate dlPFC. These results are consistent with recent data suggesting that M1R agonists may be helpful in the treatment of schizophrenia, a cognitive disorder associated with NMDAR dysfunction.

Laminar Differences in the Targeting of Dendritic Spines by Cortical Pyramidal Neurons and Interneurons in Human Dorsolateral Prefrontal Cortex.

Activation of specific neural circuits in different layers of the primate dorsolateral prefrontal cortex (DLPFC) is essential for working memory, a core cognitive function. Recurrent excitation between pyramidal neurons in middle and deep layers of the DLPFC contributes to the laminar-specific activity associated with different working memory subprocesses. Excitation between cortical pyramidal neurons is mediated by glutamatergic synapses on dendritic spines, but whether the relative abundance of spines receiving cortical inputs differs between middle and deep cortical layers in human DLPFC is unknown. Additionally, GABAergic inputs to spines sculpt pyramidal neuron activity. Whether dendritic spines that receive a glutamatergic input from a cortical pyramidal neuron are targeted by GABAergic interneurons in the human DLPFC is unknown. Using triple-label fluorescence confocal microscopy, we found that 1) the density of spines receiving an input from a cortical pyramidal neuron is greater in the middle than in the deep laminar zone, 2) dendritic spines dually innervated by a cortical pyramidal neuron and an interneuron are present in the human DLPFC, and 3) the density of spines dually innervated by a cortical pyramidal neuron and an interneuron is also greater in the middle than in the deep laminar zone. Ultrastructural analyses support the presence of spines that receive a cortical pyramidal neuron synapse and an interneuron synapse in human and monkey DLPFC. These data support the notion that the DLPFC middle laminar zone is particularly endowed with a microcircuit structure that supports the gating, integrating and fine-tuning of synaptic information in recurrent excitatory microcircuits.