Amy F. Arnsten - US grants

laboratory mouse; learning stimulant; prefrontal lobe /cortex; single photon emission computed tomography

The catecholamines, dopamine and norepinephrine, decrease in the cortex of aged individuals, and norepinephrine is further depleted in patient with Alzheimer's Disease. The proposed research utilizes aged nonhuman primates as a model system to study how norepinephrine and dopamine loss contribute to the cognitive deficits which develop with advancing age. The research focuses on the principal sulcal region of the prefrontal cortex, as this cortex is especially vulnerable to the aging process, both in its loss of catecholamines and its ability to perform cognitive functions. Intracortical infusions directly into the principal sulcal cortex, as well as systemic administration of pharmacological agents will be used to observe how catecholamines act at specific receptors to affect the working memory abilities of the prefrontal cortex in the aged primate. Dopamine's effects at D1 and D2 receptors will be explored using the newly available D1 and D2-selective agents, and interactions between these receptors will be examined through combined drug treatment. Norepinephrine's actions as alpha-1 and beta receptors will be compared to its beneficial influence at alpha-2 receptors in the prefrontal cortex, and we will explore the possibility that alpha- 2 and beta receptor stimulation interact to elevate cAMP levels in prefrontal cortex and produce long lasting improvement in working memory abilities. Finally, we will more fully characterize the cognitive effects and mechanism of action of guanfacine, and alpha- 2 agonist which we've found to improve memory without hypotensive or sedative side effects. On the basis of our findings in aged monkeys, guanfacine currently is being tested in patients with Benign Senescence, Alzheimer's Disease and Korsakoff's amnesia, thus demonstrating the immediate clinical relevance of this project.

The overall purpose of this two-part project is to examine drug interactions in human and nonhuman primates with particular reference to cognitive function. In Part A, monkeys will be the subjects of inquiry whereas in Part B, the initial studies will be conducted to normal human adults. Although higher cortical functions are disturbed in schizophrenia, little is known about how these transmitters influence cortical circuitry in the primate. The proposed research will use behavioral pharmacological techniques to examine the effects of dopaminergic, serotonergic and glutamatergic compounds on higher cortical function. Experiments in rhesus monkeys will focus on the cognitive abilities of the prefrontal, inferior temporal and parietal association cortices, as well as assessing fine and gross motor function and general behavioral changes. The ability of D1 receptor stimulation to enhance cognition will be studied using the newly available, full D1 agonist, dihydrexidine. The D2 agonist, quinpirole, has recently been found to have multiple effects on behavior, including improvement in working memory performance and the production of "hallucinatory-like" behavior. The receptor mechanisms underlying these behavioral changes will be examined using antagonists with varying affinities for cloned, dopamine receptor subtypes (eg D2 vs D3 receptors). In addition, D1 and D2 agonists will be co-administered to observe whether excessive DA stimulation can lead to cognitive dysfunction. Results with dopamine compounds will be compared to those with serotonergic drugs which act at the 5HT2 and 5HT1c receptors. Agonists at these receptors produce hallucinations in humans; they may also produce "hallucinatory-like" behavior and cognitive changes in monkeys. The receptor subtype responsible for hallucinatory-like behaviors will be identified using a new, selective 5HT2 antagonist. As many D1 compounds bind at 5HT2/5HT1c receptors, these experiments will also serve as important controls. The studies of Part B, like those planned in monkeys, will focus on tests of frontal lobe function, particularly the Wisconsin Card Sort Test, but also delayed recall of object names and verbal fluency, among other tests. These studies will examine the relative efficacy of the typical and atypical neuroleptics, haloperiodol and clozapine, to block the deficit producing effects of ketamine in healthy subjects. Comparative studies of dopamine interactions with glutamate receptors will be carried out on rhesus monkeys in Part A. These studies are expected to lead to an understanding of the pharmacological interactions in the primate prefrontal cortex that are relevant to schizophrenia.

DESCRIPTION (provided by applicant): Depression and Post-Traumatic Stress Disorder (PTSD) are more prevalent in young women than men. Genetic studies link the CREB1 gene with recurrent depression in women but not men, supporting a neurobiological basis for this gender discrepancy. Both depression and PTSD involve dysfunction of the prefrontal cortex (PFC), a brain region that is 1) critical for regulating behavior, thought and affect, and 2) dysfunctional during exposure to uncontrollable stress. Stress is a major risk factor for depression, and life-threatening stress can cause PTSD. We hypothesize that estrogen promotes sensitivity to stress, rendering women more susceptible to PFC dysfunction, and thus to stress-related disorders. The proposed research will begin to identify the neurobiological mechanisms through which estrogen exacerbates stress-induced PFC dysfunction. Research in male rats has shown that stress-induced PFC dysfunction arises from excessive catecholamine release in PFC, activating protein kinases A and C, which in turn phosphorylate CREB. Interestingly, alpha-1 adrenoceptors (al R) drive this stress response, while alpha-2A adrenoceptors (a2AR) protect the PFC from stress, and estrogen is known to increase the expression of a1R and reduce the expression of a2AR. Our initial results show that cycling female rats with high levels of circulating estrogen are impaired by mild injection stress, by low doses of a pharmacological stressor, FG7142, and by brief periods of restraint stress which have no effect in males or females with low levels of estrogen. The proposed research will extend these studies to ovariectomized rats with and without estrogen replacement. Aim 1 will test the hypothesis that estrogen amplifies the cognitive and biochemical responses to restraint stress. Biochemical characterization will include measures of a1AR and a2AR expression, catecholamine turnover and phospho-CREB in PFC. Aim 2 will begin to explore the contribution of these biochemical changes to PFC cognitive function, testing whether the increased expression of a1R in the PFC of estrogen-treated rats renders them more sensitive to cognitive impairment. This research will begin to reveal how estrogen amplifies the neurochemical cascades that lead to activation of CREB and dysfunction of the PFC during stress

Dopamine (DA) has a critical influence on the working memory and attention functions of the prefrontal cortex (PFC): Modest levels of D1 receptor stimulation enhance, while high levels impair, PFC cognitive function. The proposed research will provide the first characterization of the intracellular signaling mechanisms underlying these opposing DA actions. Direct infusions into rat PFC will reveal the contributions and interact ons between IP3/calcium signa ing via ca cyon, protein kinase C (PKC) and cAMP/protein kinase A (PKA) mechanisms. Aim 1 will determine the second messenger mechanisms underlying the cognitive enhancement induced by low doses of D1 receptor agonists, either administered systemically (similar to human subjects in Project 7), or by direct infusion into the rat PFC. We hypothesize that these enhancing effects will be mediated by calcyon/IP3 signaling, possibly amplifying internal calcium release within the dendritic tree and amplifying dendritic integration (see Project 3). Aim 2 will determine the second messenger mechanisms underlying the cognitive impairment induced by high doses of D1 receptor agonist. Preliminary results suggest that cAMP/PKA signaling mediates the impairment observed at high levels of D1 receptor stimulation, perhaps eroding information transfer from dendrite to soma. We will also test for possible interactions between signaling pathways: eg internal calcium release may potentiate detrimental PKA actions by stimulating adenylyl cyclase isoforms 1 or 8. These studies will explain why high levels of D1 receptor stimulation markedly impair PFC cognitive functioning. Finally, Aim 3 will assess the role of calcyon in PFC cognitive functioning using genetically altered mice created in Project 1. Mice with an overproduction of calcyon will be compared to wildtype controls (and possibly to mice with disabled calcyon). As schizophrenic patients have abnormally high calcyon levels in PFC, the calcyon overexpressing mice will have direct clinical re evance. Mice will be assessed on a battery of PFC vs. control tasks and subsequently tested for their response to D1 agonists. We predict that mice with overexpression of calcyon, similar to schizophrenics will show an exaggerated improvement following low doses of D1 agonists. These studies will provide the first assessment of calcyon's contribution to PFC cognitive function.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The goal of this application is to provide highly focused training in Systems Neuroscience at Yale University School of Medicine, with special emphasis on the mammalian cortex. The philosophy of this program is to preserve and foster integrative approaches to neurobiology that will interface with molecular genetics and clinical medicine with respect to development, organization, and plasticity of the mammalian brain. Forty-three faculties from 17 basic and clinical departments are participants in this multidisciplinary program. The program offers both depth and breadth. The depth derives from its unique substantive focus on cortical circuits of the rodent, primate and human brain. The breadth of the program derives from the diversity of approaches, spanning developmental, systems and cognitive neuroscience. Faculty interests span axonal guidance mechanisms in embryos to memory decline and stroke in elderly humans. Methodologies include cloning; cell culture; immunocytochemistry; in situ hybridization; electron and two photon microscopy; voltage clamp and whole cell recording; calcium imaging; biochemistry and molecular analyses; psycho-pharmacology; rodent, monkey and human behavior; in vivo extracellular recording in behaving animals; and fMRI and PET imaging in human subjects. Two predoctoral and two postdoctoral positions are requested. Trainees are selected from a variety of backgrounds in biological sciences on the basis of their potential for excellence and leadership in research by an Admissions Committee (predocs) or Executive Committee of the NCS (postdocs). Mentors are Ph.D.s and M.D.s. with NINDS grants and/or NINDS related research foci. Training includes coursework, intensive research apprentice-ship, structured seminar programs, and laboratory and departmental presentations of research progress. [unreadable] [unreadable]

NIH Roadmap Initiative tag; bioimaging /biomedical imaging; disease /disorder etiology; prefrontal lobe /cortex

Animal Core- The responsibilities of the Animal Core are 5-fold: (1) To coordinate the purchase and numbering of young and aged rats and monkeys for all four Projects;(2) To train rats and monkeys on the appropriate cognitive tasks (Projects 1, 2 and 4);(3) To perform neural implant aseptic surgeries for monkeys used in electrophysiological studies (Project 1), and rats receiving viral transfections in prefrontal cortex (Project 2);(4) To perform cognitive assessment of rats (Projects 2 and 4) and monkeys (Project 4) in order to evaluate the effects of viral transfection or chronic drug treatment;and (5) To assist with chronic drug treatment (Project 4). Cognitive testing will include several tests of spatial working memory. For Project 1, young and aged monkeys will be habituated to sitting in a primate chair while interacting with a computer monitor. They will then be trained on the oculomotor spatial delayed response task. Training aged monkeys to perform this task requires patience, but is feasible if done gradually. Projects 2 and 4 will require training of young and aged rats on the spatial delayed alternation task in a T maze. A separate group of rats will also be trained on a control task, spatial discrimination, in the same T maze, to examine the specificity of viral manipulations in prefrontal cortex on cognitive function. Finally, young and aged monkeys in Project 4 will be trained and tested on spatial delayed response in a Wisconsin General Test Apparatus, a classical test of dorsolateral prefrontal cortical function. Animals in chronic drug studies will be tested twice weekly. The experimenter testing the animals will be unware of the drug or viral treatment conditions. Cognitive data will be submitted to the Administrative Core for statistical analysis, to examine the relationships between cognitive, histological and /or biochemical data. LAY SUMMARY: The Animal Core will train and test young and aged animals on memory tasks. We will see if drugs or molecular manipulations in the brain of rats will slow the aging process and improve memory.

Stress diminishes regulatory control of behavior by the prefrontal cortex (PFC);while heightening subcortical mediation of habits. Project 2 of this consortium will examine the molecular'and cellular basis of RFC dysfunction during acute and chronic stress, with the aim of identifying hovel therapeutic targets. Previous research has found that stress impairs PFC funetibh through 1) excessive production of CAMP via dopamihe (DA) D1 and nOrepinephririe (KlE) beta! receptors, and 2) NE alpha-1-activation of phosphbtidyl inositoi (PI) DAG-prOtein kihase C (PKC) signaling, suppressing PFC cell firing. The proposed research will further explore the signaling cascades contributing to PFC dysfunction by examining the role of the IP3-Ca2+ component of PI signaling. Consistent with this possibility, in vitro recordings from PFC neurons show that rP3-mediatedihterharCa2+ release opens SKchannels thereby suppressing PFC cell excitability, the proposed research will examinei whether this rhechahisnrcontributes to stress-induced PFC dysfunction at 3 levels: Aim 1 will use in vitro recordings and Ca2+ fluorescence imaging of PFC pyramidal neurons to examine the cellular basis of the PI cascade, Aim 2 will extend these results to in vivo recordings of PFC neurons in animals performing working merhbry tasks, and Aim 3 will test whether PFC cognitive functions can be protected from stress by blocking IPS receptors or SK channels. Aim 3 will also assess agents that can be administered to humans. We will test whether blocking alpha-1 and beta Kl'E receptors with carvedilol protects PFC function from stress. If successful in animals, carvedilol can be tested in humEins exposed to stress in Project 9;We will also test the role of endbcanrtabanoids (eCB) in stres^induCed PFC dysfunction as an extension of Project 5. Because eCBs depend on DAG and Ca2+, this work is diredtly relevant to PI signaling. We will test whether pharmacological manipulation of eCB signaling with Rimbnabant ahd URB597 alters PFC physiology and cbghitibn as a prelude to possible human testing in Project 9. Finally, Aim 4 will determine whether PI signaling contributes to spine loss on PFC neurons during chronic stress. PKC phosphorylation of MARCKS disrupts actin, which may contribute tb spine loss. We will test whether Chronic PKC inhibition with Chelerythrine protects PFC neurons from spine loss. As chelerythrine is in pfeclinical development, this may provide another strategy for increasing PFC regulation of behavior in humans.

The responsibilities of the Administrative Core are to coordinate scientific collaboration within the program project, to ensure the proper transfer of tissue and evaluation of data between Projects and Cores, and to deseminate data to researchers interested in computational modeling of prefrontal cortical (PFC) circuits in the aging brain. The Administrative Core will faciltate scientific collaboration between Projects and Cores through regular lunchtime meetings. This PO1 brings together experts in cortical electrophysiology, intracellular signaling, neuroanatomy, higher cognitive function, and cortical modeling in order to tackle the problem of aging cortical circuits from a multi-disciplinary approach. We have aspired to pair junior investigators with more senior scientists to introduce new scientists to the field of aging research. The P01 is highly interactive, depending on shared resources and multi-level analysis of brain tissue. A major responsibility of the Core is integrating data between Projects and Cores. Within-subjects data from Projects 2-4 and the Histology and Animal Cores will be fed into the Administrative Core for multi-variate analysis by our statistician, Dr. DiPietro. We will determine how network strength, working memory, dendritic integrity and molecular signatures interact to alter cognitive abilities in the aging brain. The findings from this PO1 will be provided to Dr. Xiao-Jing Wang for computational modeling of aging effects on PFC networks. Dr. Wang is an expert on computational models of PFC working memory networks, examining how modulatory influences allow PFC networks to resist distraction. Thus, the data from this PO1, particularly the electrophysological findings from Projects 1 and 3, will be of immediate relevance to this endeavor. To our knowledge, this will be the first time that the effects of aging will be included in a computational model of the PFC. Finally, relevant data from this PO1 will be posted on the Senselab database for sharing with the neuroscience community. We are fortunate to have Dr. Gordon Shepherd, the founder of Senselab, as consultant to this Administrative Core. Senselab includes several databases which are linked to the Society for Neuroscience Database Gateway. Thus, the findings gained in this PO1 on the aging of PFC networks will be readily available to interested neuroscientists. LAY SUMMARY: The Administrative Core will coordinate Projects and Cores, integrate data for statistical analysis and share data with other scientists.

DESCRIPTION (provided by applicant): The working memory functions of the prefrontal cortex (PFC) decline early in the aging process. Working memory depends on recurrent excitation between PFC pyramidal cell microcircuits. With advancing age, there is a loss of pyramidal dendritic spines, thus eroding the anatomical substrate for circuit connectivity. PFC circuits may also be weakened by molecular changes that alter the functional status of network connectivity. NE (norepinephrine) strengthens PFC cognitive function through actions at post-synaptic a2A adrenoceptors (a2A-AR), which inhibit the production of cAMP. cAMP opens HCN (hyperpolarizationactivated cyclic nucleotide gated) channels on PFG dendritic spines, lowering membrane resistance and weakening the efficacy of synaptic inputs. With advancing age, there are fewer a2A-ARs, disinhibited cAMP and increased HCN channels in the PFC. The proposed research tests the hypothesis that excessive cAMP/HCN signaling underlies PFC cognitive deficits early in the aging process, and contributes to eventual spine loss through weakened synaptic connectivity. The research involves 4 Projects and 3 Cores. Project 1 will record from ensembles of PFC neurons in young vs aged monkeys performing working memory tasks to test the hypotheses that PFC networks are weakened with age, and that connectivity can be strengthened by iontophoretic application of agents that inhibit cAMP or block HCN channels. Project 2 will assess molecular changes in the aging rat and monkey PFC that may impact cAMP/HCN signaling and spine loss. This project will also use adenoviral transfection of rat PFC to test whether altered expression of HCN channels and other signaling proteins slow age-related decline in working memory and spine density. Project 3 will record from ensembles of PFC neurons in young and aged rats, and thus observe changes in circuit strength over time in response to viral (Project 2) or pharmacological (Project 4) manipulations. Project 4 will use electron microscopy to visualize changes in NE axons, a2A-AR, and HCN channels in the aging PFC, and will test whether chronic stimulation of a2A-AR with guanfacine will slow working memory impairment and spine loss in aging rats and monkeys. As guanfacine is available for human use, this research can readily translate to treating PFC deficits in the elderly.

Project 4- The cognitive functions of the prefrontal cortex (PFC) are impaired early in the aging process, and there is significant spine loss from the dendrites of aged PFC neurons. Project 4 will examine whether these age-related changes can be prevented by chronic treatment with the alpha-2A noradrenergic (NE) agonist, guanfacine. We hypothesize that guanfacine will inhibit detrimental cAMP actions in the PFC that lead to weakened connectivity in PFC networks. Electron microscopy (EM) studies of young PFC have shown that alpha-2A receptors are co-expressed wih HCN (Hyperpolarization-activated Cyclic Nucleotide-gated) channels in the spines of monkey PFC neurons, where they are ideally positioned to gate synaptic events. When the HCN channels are opened by cAMP, they weaken inputs onto the spine. Guanfacine inhibits the production of cAMP and strengthens functional connectivity. With advancing age, there is a decrease in alpha-2 receptors, evidence of excessive cAMP signaling, and increased HCN expression. We hypothesize that disinnibited cAMP actions early in the aging process weakens the functional connectivity of PFC networks, contributing to declines in PFC cognitive function. With advancing age, sustained synaptic weakness would lead to spine loss. Aim 1 will test this hypothesis by examining ultrastructural changes in dendrites, NE axons, alpha-2A receptors, and HCN channels in the superficial layers of PFC from young, middle aged and aged monkeys. We will use dual immunoEM to visualize the distribution of these molecules, and the integrity of dendrites, with particular focus on layer I. We predict that there will be changes in neuromodulators early in the aging process (e.g. decreased NE axons and alpha-2A receptors in layer I, increased HCN channels on dendritic spines), followed by architectural decreases in dendritic spines with advancing age. Aims 2 and 3 will examine whether chronic stimulation of alpha-2A receptors with guanfacine will reduce functional weakening of synapses and thus prevent spine loss and working memory decline in aging rats and monkeys. Preliminary results indicate that chronic guanfacine produces enduring improvements in spine density and working memory abilities. As guanfacine is available for human use, this research is directly relevant to the treatment of age-related cognitive decline. LAY SUM: The research will test whether chronic treatment with guanfacine will protect brain cells and memory in aged animals.

DESCRIPTION (provided by applicant): Cognitive disorders such as Alzheimer's Disease (AD), Fronto-Temporal Dementia and schizophrenia are a tremendous burden on our society, as patients are often unable to care for themselves, and require extensive resources for many years. These disorders will be an even greater burden as our society grows older in the next decades. Current treatments are inadequate, and research in this arena continues to focus on mouse models. However, AD, schizophrenia, and related cognitive disorders primarily afflict the highly evolved association cortices which are poorly developed in mice, while the primary sensory cortices are little affected in these disorders. What makes the association cortices so vulnerable? And why are more basic cortical areas, such as the sensory cortices, more resistant to disease? These are fascinating evolutionary questions with immediate medical relevance. The proposed research will test the hypothesis that the highly evolved primate association cortices are more vulnerable to disease because they are regulated by Ca2+-cAMP signaling pathways in a fundamentally different manner than the evolutionarily older, sensory cortices, and that dysregulation of Ca2+-cAMP signaling following genetic or environmental insults predisposes these higher circuits to dysfunction and degeneration, e.g. through hyper-phosphorylation of tau. Our data have revealed that primate prefrontal association circuits contain high levels of cAMP-regulated K+ channels near their network connections that normally serve to gate inputs and provide mental flexibility. However, this process requires precise regulation, and even small insults to regulatory processes impair cognition and may increase risk for degeneration. A striking number of these proteins are genetically linked to schizophrenia, and show changes with advancing age. We hypothesize that primate cortical circuits will have differing sensitivities to Ca2+-cAMP signaling based on their evolutionary st

DESCRIPTION (provided by applicant): The proposed research will examine whether metabotropic glutamate receptors (mGluR) have potential as therapeutic targets for treating age-related cognitive decline. The work will reveal mGluR actions in the evolved primate circuits that subserve the higher cognitive functions impaired by normal aging in monkeys and humans. The choice of drugs was advised by Dr. Jeffrey Conn, a world expert in mGluR pharmacology. We will examine how mGluR2/3 and mGluR5 agents influence prefrontal cortical (PFC) physiology and cognition in young vs. aged monkeys. This research is timely, as mGluR5 antagonists are being developed for Fragile X Syndrome, while mGluR2/3 agonists are being developed for schizophrenia. Thus, these compounds may have off-label use in the elderly to normalize calcium (Ca2+) and cAMP signaling in the aged PFC. A variety of data have shown evidence of increased Ca2+-cAMP signaling in the aged PFC, which reduces neuronal firing by opening Ca2+- and cAMP-regulated K+ channels on PFC synapses. mGluR5 and mGluR2/3 are localized both pre- and post-synaptically on highly evolved, layer III synapses in primate PFC. It is generally appreciated that mGluR5 antagonists and mGluR2/3 agonists can decrease excitotoxicity by pre-synaptic inhibition of glutamate release. However, these agents may also have beneficial actions at post-synaptic receptors in the primate PFC, reducing intracellular Ca2+ release and inhibiting cAMP signaling, respectively. Aim 1 will characterize the effects of mixed mGluR2/3 compounds (the agonist, (2R,4R)-APDC vs antagonist, LY341495) on Aim 1A) working memory and recognition memory performance, and Aim 1B) PFC neuronal firing, in young vs. aged monkeys to see if mGluR2/3 agonists may have potential as cognitive enhancers for the elderly. Aim 2 will begin to dissect mGluR2 vs. mGluR3 actions in primate dlPFC using newly available, selective mGluR2 vs. mGluR3 compounds, examining their effects on Aim 2A) cognitive performance and Aim 2B) neuronal firing. Aim 2C will also use immunoelectron microscopy (EM) to determine the pre- vs. post- synaptic location of mGluR2 vs. mGluR3 in highly evolved primate cortical circuits, their interactions with Ca2+- cAMP-K+ channel signaling proteins in PFC synapses, and any changes in distribution with advancing age. Aim 3 will characterize mGluR5 actions in primate dlPFC. These studies will examine the effects of the selective mGluR5 agonist, VU0360172, vs. the selective mGluR5 antagonists, MTEP or fenobam, on: Aim 3A) working memory and recognition memory abilities, and Aim 3B, PFC neuronal firing, in young and aged monkeys. Aim 3C will use immunoEM to localize mGluR5 in the young vs. aged primate cortex to see if mGluR5 co-localize with Ca2+-cAMP- K+ channel signaling proteins in spines, and whether there are any age- related changes in the pre- vs. post-synaptic distribution of these receptors. Preliminary data show improved cognitive performance and enhanced neuronal firing with low doses of the mixed mGluR2/3 agonist, APDC, or the mGluR5 antagonist, MTEP, in aged monkeys, consistent with potential therapeutic actions.

DESCRIPTION (provided by applicant): The proposed research will provide the first study of metabotropic glutamate receptor mGluR2/3 influences on dorsolateral prefrontal cortex (dlPFC) function in primates, a brain region greatly afflicted in patients with schizophrenia. Group II metabotropic glutamate receptors (mGluR2, mGluR3) are potential therapeutic targets, and genetic alterations of mGluR3 are increasingly associated with schizophrenia. However, there has been no research on how these receptors influence dlPFC circuits in primates, where there are likely evolutionary differences. The proposed research will determine the pre- vs. post-synaptic location of mGluR2 vs. mGluR3 in primate dlPFC circuits, and how mGluR2/3 stimulation influences dlPFC neuronal firing and cognitive function. As selective mGluR2 and mGluR3 agents are now available for research use, the proposed research will begin to dissect mGluR2 vs. mGluR3 effects on neuronal firing and cognitive performance. Preliminary data show that low doses of an mGluR2/3 agonist can enhance dlPFC neuronal firing and improve working memory performance in monkeys, with no evident side effects, suggesting clinical potential. Aim 1 will use multiple label, immunoelectron microscopy to localize mGluR2 and mGluR3 in pre- vs. post-synaptic sites in the primate dlPFC. Although rodent studies have focused on pre-synaptic localization, preliminary data from primate dlPFC indicate that mGluR2/3 are also localized post-synaptically next to layer III excitatory synapses, positioned to strengthen network firing. Localization in dlPFC will be compared to that in orbital PFC, temporal cortex and primary visual cortex, to see if the pattern in dlPFC is unique, or extends to other association and/or sensory cortices. Aim 2 will perform single unit recording of dlPFC neurons in monkeys performing a spatial working memory task to observe how alterations in mGluR2/3 signaling influence task-related network firing. These studies will be able to observe whether a drug treatment decreases neuronal firing (consistent with pre- synaptic inhibition of glutamate release), or increases neuronal firing (consistent with post-synaptic inhibition of cAMP-K+ channel actions). Results with mixed mGluR2/3 compounds will be compared to newly available, selective mGluR2 and mGluR3 agents to begin to define specific influences on dlPFC neuronal firing. Preliminary data indicate that low doses of an mGluR2/3 agonist greatly enhance the firing of dlPFC Delay cells that maintain working memory. Aim 3 will characterize the behavioral effects of the compounds used in Aim 2, in monkeys performing a battery of cognitive tasks. Initial data indicate that low doses of the mGlu2/3 agonist, (2R,4R)-APDC, markedly improve working memory performance with few side effects, highlighting the therapeutic potential of these mechanisms. The proposed experiments will compare the effects of mGluR2/3 compounds to those of selective mGluR2 and mGluR3 agents to see which receptor(s) underlie the cognitive enhancement. These data will provide essential information for guiding therapeutic strategies for cognitive enhancement in schizophrenia, and for understanding how insults to mGluR3 can impact cognitive function.

? DESCRIPTION (provided by applicant): The NMDA receptor (NMDAR) antagonist, ketamine, is currently in experimental use for the treatment of severe, intractable depression. Emerging clinical data indicate that intranasal ketamine administration can produce relief within minutes (5~40min), with fewer cognitive side effects than systemic ketamine injections. The proposed research will use a non-human primate paradigm to study the cellular and circuit mechanisms underlying the effects of intranasal vs. injected ketamine administration in prefrontal cortical (PFC) circuits immediately relevant to depression and cognition. We have observed persistent neuronal activity in medial PFC related to aversive events in a decision-making task, which may contribute to negative affective states and be abnormally heightened in depression. We will test the hypothesis that ketamine rapidly erodes the persistent representations of loss and punishment generated by medial prefrontal neurons, similar to ketamine's ability to erode persistent representations of visual space by dorsolateral prefrontal cortical (dlPFC) neurons. We hypothesize that the disruption of neural circuits representing loss may underlie the ultra-rapid effects of intranasal ketamine in patients (within minutes), while spinogenesis in higher order PFC regions may contribute to the more sustained anti-depressant actions (hours to days). As intranasal inhalation delivers drug directly to the brain through the holes in the cribiform plate, we further hypothesize that the medial PFC regions in the direct trajectory of intranasal ketamine may be more affected than the dlPFC neurons that are more distant. Such data would help to explain why intranasal administration has a more rapid onset with fewer cognitive side effects than ketamine injections. Aim 1 will compare the effects of intranasal vs. intramuscular administration of sub-anesthetic doses of ketamine on performance of the decision-making vs. spatial working memory tasks. We predict that ketamine will attenuate the effects of previous punishment on decision-making, allowing a more resilient behavioral response, and that intranasal administration will preferentially influence this behavior, while IM injections will have more global effects on performance in both tasks. Aim 2 will compare the effects of intranasal vs. intramuscular administration of ketamine on persistent neuronal firing in medial PFC regions relevant to depression (BA24, BA25 and dmPFC) compared to the dlPFC. We predict that ketamine will rapidly erode persistent representations of loss in medial PFC areas. We further hypothesize that intranasal administration will have more targeted effect on medial PFC neurons, while IM ketamine will alter neuronal firing in both medial and lateral PFC areas. Finally, Aim 3 will test whether the effects of ketamine are mediated by local actions in the medial PFC using iontophoretic application of ketamine and other more selective NMDAR NR2A vs. NR2B antagonists directly onto medial PFC neurons, similar to previous experiments in the dlPFC. The results from these experiments will provide important insights into the cellular and circuit mechanisms underlying the rapid anti-depressant effects of ketamine.

The proposed research will test a novel mechanism for protecting higher cognition and reducing tau phosphorylation in the aging cortex, based on the unique needs of the newly evolved association cortical neurons most vulnerable in Alzheimer's Disease (AD). These circuits are powerfully modulated by feedforward, cAMP-PKA-calcium signaling, which becomes dysregulated with advancing age, leading to: 1) excessive opening of K+ channels, reducing neuronal firing and impairing cognition, and 2) phosphorylation of tau. Early stages of tau phosphorylation by PKA can be seen in aged rat association cortex, while more advanced stages are seen in aged monkey cortex, including fibrillated tau labeled by the AT8 antibody, and accompanying A? expression. The proposed research will test a strategy to regulate cAMP-PKA-calcium signaling to reduce AD- like pathology in the aging cortex by amplifying the brain's natural protective actions at glutamate metabotropic receptors type 3 (mGluR3). mGluR3 have neuroprotective actions on astrocytes, and additional, regulatory actions on neurons. New data have revealed post-synaptic mGluR3 in prefrontal association cortex (PFC) that regulate cAMP-PKA-calcium signaling. The proposed research will enhance stimulation of mGluR3 by its endogenous ligand, NAAG (N-acetylaspartyl-glutamic acid), via inhibition of the enzyme that destroys NAAG, GCPII (glutamate carboxypeptidase II). GCPII inhibitors are under development for treating inflammatory disorders, with excellent tolerability and minimal side effects in phase I human testing. We will observe whether mGluR3 are positioned to regulate cAMP-PKA phosphorylation of tau in the primate entorhinal cortex (ERC) and PFC circuits most vulnerable in AD, and whether GCPII inhibition can restore neuronal firing, improve cognitive function, inhibit phosphorylation of tau, and reduce neuroinflammation in aged rats and monkeys. This work will confirm and extend the efficacy of two structurally distinct GCPII inhibitors, 2-MPPA and 2- PMPA, under development by the Johns Hopkins Drug Discovery group. Aim 1 will use dual immunoelectron microscopy to confirm that mGluR3 are correctly positioned to regulate cAMP-PKA phosphorylation of tau (pS214Tau) and AT8-labeled tau in aging ERC and PFC glutamatergic synapses. Aim 2 will test whether acute administration of 2-MPPA or 2-PMPA enhances memory-related PFC neuronal firing in aging monkeys by regulating cAMP-PKA opening of K+ channels, and whether systemic administration can improve cognition in aged rats and monkeys with minimal side effects. Aim 3 will test whether chronic treatment with an optimal dose of 2-MPPA produces sustained improvement in working memory, and reduces tau phosphorylation and neuroinflammation in aged rat and monkey association cortex. We will have the rare opportunity to see if chronic 2-MPPA treatment in aged monkeys reduces both fibrillated AT8-labeled tau, and A? expression. Preliminary data indicate that these agents produce very robust improvements in cognition with no evidence of side effects, encouraging a therapeutic strategy with feasible translational application.

This award provides support for the US component of a NeuroNex Network consisting of 15 research teams in the US, Canada, and Germany. This consortium will address the molecular, cellular, and circuit mechanisms underlying working memory in primates. This is fundamental to abstract thought and cognitive ability. The central hypothesis to be tested is that there is an evolutionarily driven expansion of visual connectivity in different regions of the brain and along the visual processing pathway, and that this connectivity principal will be more pronounced in macaques than marmosets. The research targets three regions, reflecting three levels of the visual pathway. A long-lasting, far-reaching impact involves leveraging work from this NeuroNex Network with other BRAIN Initiative projects to enable acquisition and sharing of the new knowledge. Future applications, even beyond the brain, of the knowledge and tools developed here will give rise to data that address fundamental and novel principles of complex self-organizing systems. The NeuroNex Network also involves training the next generation, including through inter-laboratory and fellow exchanges.

This NeuroNex Network integrates teams of four Interdisciplinary Research Groups (IRGs). The approach involves characterizing neurons in each of three regions of the dorsolateral prefrontal cortex (LPFC) and processing stage using genotyping and transcriptomics, and then examining linkages to basic electrophysiological properties. These areas also represent three stages of information processing along the primate cortical pathways as well as in the evolution of cortical layers. It is hypothesized that the functional, anatomical and molecular dependencies of neurons vary across these regions, and that differences are prominent in macaques, and more subtle in marmosets. The data are to be used to drive the development of computational models. The first IRG takes an in vivo physiology approach using laminar recordings. The second IRG addresses in vitro electrophysiology and neuronal circuitry. The third IRG is a molecular characterization using transcriptomics and immuno electron microscopy. The fourth IRG takes a neuroinformatics approach that combines these data to inform computational models of cortical architectures that mimic the single neurons and population dynamics measured in the first IRG. This IRG will also create a centralized resource to assess data across levels and IRGs. This project is co-funded by Emerging Frontiers in the Directorate for Biological Sciences and the Cyberinfrastructure for Emerging Science and Engineering Research (CESER) program within the Office of Advanced Cyberinfrastructure.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary The goal of this research proposal is to develop brain-penetrant inhibitors of glutamate carboxypeptidase II (GCPII) as a new therapeutic strategy to improve cognition and reduce risk of late-onset Alzheimer's Disease (AD). GCPII (EC 3.4.17.21) is a membrane-bound zinc metallopeptidase that cleaves the C-terminal glutamate from N-acetylaspartylglutamate (NAAG) producing N-acetylaspartate and glutamate. NAAG is known to act as an endogenous agonist at metabotropic glutamate receptor type 3 (mGluR3) and we have recently found that NAAG can enhance memory-related neuronal firing in monkeys through stimulation of Gi/Go-mediated regulation of postsynaptic cAMP-PKA-calcium signaling. Therefore, GCPII inhibition may offer a new therapeutic approach to the cognitive impairments by increasing extracellular NAAG levels and controlling cAMP-PKA-calcium signaling dysregulated in the aging brain. In the absence of mGluR3-selective agonists and positive allosteric modulators, this approach is particularly attractive as a number of structurally diverse and potent GCPII inhibitors have been developed and preclinically evaluated in a variety of neurological disorders with a robust efficacy and an excellent side effect profile. Indeed, our preliminary data show cognitive enhancement upon treatment with 2-MPPA, a clinically tested GCPII inhibitor, in aged rats and monkeys. To date, however, efforts on clinical translation of GCPII inhibitors have been substantially limited despite the significant therapeutic potential. This prompted us to propose a broad range of pharmacological approaches to the development of brain-penetrant GCPII inhibitors. We are poised to seize this therapeutic opportunity for the treatment of age-related cognitive disorders by executing the following three Specific Aims: (Aim 1) Design and synthesis of GCPII inhibitors and their prodrugs; (Aim 2) Evaluate the pharmacokinetic (PK) profile of GCPII inhibitors in rats and monkeys; (Aim 3) Assess the effects GCPII inhibitors on cognitive function in aged rats and monkeys. The successful execution of this project will lead to a novel therapeutic strategy with greater feasibility for clinical translation to address the main healthcare needs of the growing elderly population.