Richard B. Mailman - US grants

This proposal will investigate the relationship between metabolism and neuropharmacology of three structurally dissimilar phenothiazine antipsychotic drugs [thioridazine (Mellaril), trifluoperazine (Stelazine), and chlorpromazine (Thorazine)]. The primary goal of the neuropharmacological experiments is to compare the antidopaminergic activity of the parent phenothiazines with their major metabolites. The in vitro techniques to be used include various radioligand binding assays (e.g., with [3H]-spiperone and [3H]-dopamine), and the ability of drugs to affect dopamine-stimulated adenylate cyclase activity. Another in vitro measure to bridge the gap between data from the intact animal vs. that from brain homogenates, are effects of these drugs on pre- and post-synaptic receptors modulating the release of dopamine and acetylcholine from slices of striatum or other regions. In vitro effects on other neurotransmitter systems (e.g., [3H]-QNB or [3H]-WB-4101 binding) will also be examined. In vivo studies will determine the ability of the drug metabolites or their parent compounds to inhibit amphetamine- or apomorphine-induced behaviors (e.g., stereotyped behavior, aggression, and locomotion) and to alter biochemical estimates of the activity of dopamine neurons using neurochemical methods to evaluated DOPAC, HVA, 1-DOPA, etc., as required. Both intracerebroventricular and peripheral routes of drug administration will be used in the in vivo experiments. Finally, the effects of withdrawal from chronic drug administration will be measured to determine if behavioral and neurochemical changes observed are a function of the metabolites formed from the parent compound. These functional and biochemical data relevant to these phenothiazines and their metabolites will be compared to similar studies with a classical non-phenothiazine neuroleptic (haloperidol) and a compound, SCH 23390, purported to be a specific D1 dopamine antagonist. Mechanisms of metabolism will be determined, focusing on two distinct enzyme systems, the flavin-containing monooxygenase (EC1.14.13.8) and cytochrome P450 monooxygenases. The relative role of the P450 and FCM enzymes in the bioformation of pharmacologically active metabolites, and the effects of chronic administration of these drugs on enzyme activity, will be determined. Species differences in enzyme activity will be exploited by using a species (e.g., hamsters) that should produce patterns of metabolites in blood that are similar to humans and unlike rats.

These neuropharmacological studies application will study the biochemical and molecular mechanisms by which the D1 subclass of dopamine receptors cause their psychopharmacological effects. Several hypotheses will be tested: that there is biochemical heterogeneity of D1 receptors; that only a subset of these receptors are linked biochemically to cAMP synthesis; that a subset of these D1 dopamine receptors interact functionally with D2 receptors as part of the same multimolecular complex; and that it is possible to model this receptor class at the molecular level and to use such models to design drugs that have selectivity for certain subpopulations of D1 receptors. These hypotheses will be tested by studying the basis for apparent multiplicity of D1-like receptors, e.g., by comparing the occurrence of [3H]-SCH23390 binding sites versus dopamine-sensitive adenylate cyclase activity (DA-ACase) or the potency of selected drugs to cause dopaminergically-mediated behaviors. Lesioning and pharmacological studies will be used to compare limbic areas to striatum on the basis of these functional and receptor characteristics. Several series of rigid and semi-rigid dopamine agonists and antagonists will be compared using both in vivo and in vitro pharmacological methods. These data will be used in computer-assisted molecular modeling studies to model the active site of D1 receptors. Although initially these studies will assume a single receptor, it is assumed that the biological studies ultimately will provide data permitting at least two types of sites (e.g., one linked to stimulation of adenylate cyclase, and one not) to be modeled and defined. The molecular modeling studies will be modified continually to incorporate the data from receptor solubilization, purification, and characterization studies which will be a major emphasis of this research. As a consequence of our demonstration that SCH23390 binds tenaciously to its physiologically important receptor(s), the purification experiments will rely heavily on affinity chromatography using a 4-alkylphenyl-substituted analog of SCH23390 that we will synthesize. The availability of purified or partially purified receptor(s) will lead to raising of antibodies against these proteins, and to the initiation of molecular biological study of these proteins. We will perform immunohistochemical localization of these receptors, and also conduct immunoneutralization studies. The molecular biological studies will be aimed at the physiological mechanisms involved in expression and regulation of D1 receptors (e.g., synthesis, posttranslational modifications, sites of expression, etc.).

Many environmentally important pollutants, including certain metals and their organic derivatives are neurotoxic. The development of the nervous system is influenced by extrinsic factors, and the perturbations caused by toxic metals often may be different in the developing nervous system than in the mature nervous system. We have previously demonstrated that lead has unique intoxicating effects in the immature nervous system: a persistent abnormal polydipsia to lithium of central nervous system origin; a defect in synaptogenesis lasting to senescence; and a persistent reduction in myelin. We will continue to utilize a multidisciplinary approach to ellucidate the neurotoxic effects of heavy metals, to investigate how such neurotoxic alterations affect the subsequent structural, chemical and functional development of the nervous system, and to determine the mechanisms of the neurotoxic action of selective toxic metals. A selected series of organometal derivatives will be used to investigate structure-neurotoxicity relationships. These studies will be pursued by investigating the uptake, distribution and fate of the neurotoxins and by determining the effect of these agents on the development and stability of neurotransmitter systems, axonal transport and myelination, and selected aspects of transport systems of the blood-brain barrier. Emphasis will be placed on those animal and in vitro models which most directly allow testing of the hypotheses that the developing nervous system is more sensitive to these agents and that consequences of such perturbations of neural development may be unique or tardive in their manifestations. Consistent with this, selected aspects of lead neurotoxicity will be investigated further. In addition, new studies will be pursued based upon our Program experience, especially those which offer rational comparisons with our earlier studies with lead. In this regard, initial plans call for examination of a selected series of organotin compounds and organolead compounds.

This application seeks to determine the nature and mechanisms of developmentally mediated perturbations of the primate immune, nervous, and neuroendocrine systems, caused by low doses of lead. The proposed studies seek to 1) extend, in a nonhuman primate species, earlier observations made in the laboratory rat and 2) test the hypothesis that early, low-level lead exposure will alter immune, neuroendocrine and neurochemical function. A particular strength of this proposal is the availability of a population of monkeys (Macaca fasicularis) whose blood-lead levels and age at exposure are similar to those of rodents used in independently-funded research. In addition, early lead exposure in these monkeys has resulted in subtle but significant behavioral changes in cognitive function. The experimental subjects are 20 monkeys which were dosed orally from birth with 1.5 mg/kg/day of lead using one of four (n=5) dosing regimes: Group 1, vehicle only; Group 2, lead from birth onward; Group 3, lead from birth to 400 days of age and vehicle thereafter; Group 4, vehicle from birth to 300 days of age and lead thereafter. One hypothesis, based on our rodent data, is that monkeys will show similar permanent changes in drinking behavior, as well as increases in dipsogenic effects of lithium. This effect is hypothesized to be due to alterations in dopaminergic function, particularly in lateral hypothalamus, amygdala and/or globus pallidus. A second hypothesis supported by the literature is that low doses of lead alter immune, and possibly, neuroendocrine function. A final hypothesis is that alterations in non-spatial discrimination reversal tasks are due, in part, to alterations in specific cholinergic and/or dopaminergic pathways. These hypotheses will be tested by first determining, in vivo, if the lead- treated monkeys have alterations in discrete components of water consumption, before or after lithium administration. Cell-mediated immunity will be studied by characterizing immunological cell phenotype and conducting blastogenic assays following mitogen stimulation. Neuroendocrine function will be examined by radioimmunoassay of stress- related hormones. Following these in vivo studies (year 01), the monkeys will be sacrificed, and in vitro experiments will be conducted on post- mortem tissue. Neurochemical studies will focus on selected central monoaminergic pathways measuring the concentration of monoamines and their metabolites in microdissected brain nuclei. Receptor binding studies (using quantitative autoradiography and homogenate assays) will evaluate potential alterations in the density and affinity of monoamine and cholinergic receptors. After determination of the chemical and anatomical locus of lead-induced changes, morphological studies using immunocytochemical techniques will be initiated. Because these studies will occur concomitantly with independently funded studies in rats, comparisons across genera will be possible, as will testing of new hypotheses derived from the rodent studies. Finally, careful archiving of monkey brains will permit future testing of post hoc hypotheses by us or other investigators.

This application seeks to determine the nature and mechanisms of developmentally mediated perturbations of the primate immune, nervous, and neuroendocrine systems, caused by low doses of lead. The proposed studies seek to 1) extend, in a nonhuman primate species, earlier observations made in the laboratory rat and 2) test the hypothesis that early, low-level lead exposure will alter immune, neuroendocrine and neurochemical function. A particular strength of this proposal is the availability of a population of monkeys (Macaca fasicularis) whose blood-lead levels and age at exposure are similar to those of rodents used in independently-funded research. In addition, early lead exposure in these monkeys has resulted in subtle but significant behavioral changes in cognitive function. The experimental subjects are 20 monkeys which were dosed orally from birth with 1.5 mg/kg/day of lead using one of four (n=5) dosing regimes: Group 1, vehicle only; Group 2, lead from birth onward; Group 3, lead from birth to 400 days of age and vehicle thereafter; Group 4, vehicle from birth to 300 days of age and lead thereafter. One hypothesis, based on our rodent data, is that monkeys will show similar permanent changes in drinking behavior, as well as increases in dipsogenic effects of lithium. This effect is hypothesized to be due to alterations in dopaminergic function, particularly in lateral hypothalamus, amygdala and/or globus pallidus. A second hypothesis supported by the literature is that low doses of lead alter immune, and possibly, neuroendocrine function. A final hypothesis is that alterations in non-spatial discrimination reversal tasks are due, in part, to alterations in specific cholinergic and/or dopaminergic pathways. These hypotheses will be tested by first determining, in vivo, if the lead- treated monkeys have alterations in discrete components of water consumption, before or after lithium administration. Cell-mediated immunity will be studied by characterizing immunological cell phenotype and conducting blastogenic assays following mitogen stimulation. Neuroendocrine function will be examined by radioimmunoassay of stress- related hormones. Following these in vivo studies (year 01), the monkeys will be sacrificed, and in vitro experiments will be conducted on post- mortem tissue. Neurochemical studies will focus on selected central monoaminergic pathways measuring the concentration of monoamines and their metabolites in microdissected brain nuclei. Receptor binding studies (using quantitative autoradiography and homogenate assays) will evaluate potential alterations in the density and affinity of monoamine and cholinergic receptors. After determination of the chemical and anatomical locus of lead-induced changes, morphological studies using immunocytochemical techniques will be initiated. Because these studies will occur concomitantly with independently funded studies in rats, comparisons across genera will be possible, as will testing of new hypotheses derived from the rodent studies. Finally, careful archiving of monkey brains will permit future testing of post hoc hypotheses by us or other investigators.

This research seeks to develop an understanding of the factors involved in ligand recognition by dopamine receptors, and the functional consequences of such interactions. New drugs, one product of these efforts, will be used to develop an better understanding of the functional consequences of dopamine receptor occupation in both model in vitro systems, and the mammalian nervous system. To do this we shall characterize antagonist ligand interactions with sub-populations of D1-like dopamine receptors, and develop new subclass selective antagonists. Molecular modeling studies, combined with selected syntheses, will help elucidate the structural features that give ligands potency for D1-like receptors, and lend antagonist characteristics to them. These studies will test specific hypotheses about the pharmacophore for D1 -like receptors in 3D space, with an initial focus on rigid compounds in which the "accessory aromatic" system is in the orthogonal configuration we hypothesize is important for antagonist activity at D1-like (e.g., D1 and D5) receptors. Biological data from both brain preparations and molecular expression systems will provide the input data for quantitative structure and activity studies based on computerized molecular modeling. The mechanism(s) and functional consequences of our full agonist dihydrexidine (DHX) will be compared to available partial or complex agonists (e.g., SKF 38393 or SKF 82958). The factors that mediate desensitization will be studied using clonal cell lines and stable expression systems. Hypotheses from such -work will be tested in more complex systems, using both in vitro (e.g., slice perfusion) and in vivo (e.g., behavioral and microdialysis) techniques. Additional efforts will be aimed at developing and characterizing bioavailable prodrugs of DHX (e.g., methylenedioxyDHX) or its analogs. Finally, we shall design and synthesize D1-like ligands that are "biodetectable". This will include synthesizing and characterizing radiolabeled derivatives of specifically interesting drugs (initially 3H-DHX), and also to design and synthesize fluorescent D1-ligands. Finally, Although the focus of our work has been on D1-like receptors, serendipitous findings have led to a second focus on D2 like (e.g., D2, D3, D4) receptors. Based on recent preliminary data, we shall determine if certain tetrahydrobenzophenanthridine derivatives have selectivity for D2-like post-synaptic receptors. This will involve in vitro and in vivo studies using mammalian preparations, and later, examining these drugs in systems expressing only one molecular form of this class of receptors.

training; computer system design /evaluation; computers; biomedical facility; computer system hardware; computer network; computer program /software; computer center; computer graphics /printing; mental retardation; mental health facility;

The primary goal of the Analytical and Applied Neuroscience (AAN) Core is to be the bridge (both technically and theoretically) between clinical hypotheses and the basic neuroscience and analytical parameters required for their testing. Thus, the AAN Core provides MHCRC scientist with quantification of any analyte (endogenous or exonobiotic) that will assist in conducting an approved study or the development of a preclinical research strategy that will address the investigator's hypotheses. Moreover, this Core provides these services in conjunction with consultation about the strengths and limitations of such methods and strategies for the desired studies. The services provided are cost-efficient, and often represent assistance that the investigator either could not readily obtain, or could obtain only with extreme difficulty or expense. The core uses diverse methodologies [ranging from radioimmunoassays and High Performance Liquid Chromatography (HPLC), to HPLC-electrospray mass spectrometry (HPLC-ESMS)] to quantify such molecules that are critical to the testing of scientific hypotheses, both clinical and basic. As noted above, not only does the core implement or develop the necessary techniques and perform the assays, but it also provides consultation to clinical investigators related to study design (e.g., utility of measures, sample acquisition and storage, interpretation, etc.). Finally, with approval of the MHCRC Scientific Review Committee, this core provides analytical service and collaboration to MHCRC-associated basic scientists, and by so doing, solidifies the vital link between clinic and basic scientists and their exchange of ideas. It is the range of the services provided by the core that led to its present name. The core also provides technical assistance and resources to basic MHCRC scientists for procedures and services that their work requires, but for which they have neither sufficient expertise nor means. The key to accomplishing these ends is the ability of this core to provide state-of- the-art analytical assays of both endogenous molecules and xenobiotics (and their metabolites). During the past five years, the MHCRC has taken several steps to maximize the efficiency and responsiveness of achieving these ends. Yet it is imperative to note that the AAN Core is a single unit that provides services that were formerly the domain of three separate laboratory-based cores which had been approved in the last competing application (see below). The background and rationale for this significant change in organization are discussed in detail in Section C.1.

computer center; computer system design /evaluation; biomedical facility; Internet; computer system hardware; computer network; child psychology; mental retardation; training; computer program /software; computer graphics /printing;

DESCRIPTION(Adapted from applicant's abstract): This is a revised application based on data showing that the first full D1 dopamine agonist dihydrexidine that we developed caused profound acute antiparkinsonian effects in primates. Recent data indicate that some, but not all, full D1 agonists produce marked tolerance when administered repeatedly. We also have shown that D1 dopamine receptor agonists of similar efficacy can differ dramatically in desensitization liability. Using a series of rigid Dl agonists, we shall determine if selective conformations are promoted by different full Dl agonists, and whether these conformations represent different, if overlapping, receptor states compared to those that promote G protein coupling. First, we shall study differences in how novel D1 agonists functionally desensitize hemagglutinin (HA)-tagged human D1 receptors (HA-D1R) in C-6 glioma cells as affected by the extent and/or pattern of GRK activity. The rate and extent of D1 receptor phosphorylation by different agonists, and the effects of dominant negative GRK mutants, will be determined. Molecular and pharmacological tools will be used to assess the relative roles of endogenous GRKs and PKA in such desensitization. We also shall determine the cellular response of GRK to agonist exposure by assessing its translocation and interaction with G-beta-gamma complements. Second, we shall map the phosphorylated residues of the hD1R receptor. Patterns of agonist-induced receptor phosphorylation induced by GRKs and second-messenger-kinases (e.g., PKA) will be assessed by mutating subsets of serines and threonines in HA-hDl-C-6 cells. We also shall overexpress HA-D1R with and without various GRKs in HEK293 cells. The phosphorylated HA-hD1 receptor will be isolated by immunoprecipitation, cleaved with protease, and sequenced by MALDVToF, Ion Trap, and/or nano-ESI-mass spectrometry. Finally, we shall examine the relationship between GRK activity and arrestin binding in the initiation of G protein uncoupling and functional desensitization in the HA-hD1 C-6 system. The complement of Q-arrestins will be determined, and alterations in high affinity binding of GRKs and arrestins to D1 receptors measured following exposure to select D1 agonists. The loss of receptor-G-protein coupling from binding of labeled GTP-analogs will be assessed to correlate receptor uncoupling with levels of )-arrestin binding. The necessity of 5-arrestin(s) in functional desensitization of the HA-hD1 receptor in C-6 cells will be studied using dominant-negative mutants, and the relation between GRK and p-arrestins assessed using tools developed in Aim 1 studies. These studies of beta-arrestin binding and recruitment will provide another functional endpoint in the delineation of the molecular mechanisms of D1 receptor desensitization.

DESCRIPTION (Adapted from applicant's abstract): The foundation of this FIRST Award is the testing of a novel concept termed the "functional selectivity hypothesis." It posits that the interaction of "atypical" drugs with a single G-protein receptor isoform may cause functional effects as extreme as agonist vs. antagonist (depending on the nature of the involved G-proteins and the type of conformational changes induced by drug-receptor interaction). While this hypothesis is proposed to generalize to all G-protein coupled receptor systems, I propose to test and refine this hypothesis by studying D2-like dopamine (DA) receptors. The first data in support of this notion were result showing that novel hexahydrobenzo.a.phenanthridine dopaminergic ligands [i.e., dihydrexidine (DHX) and its analogs] activated post-synaptic, but not pre-synaptic, D2-like receptors. The current application will focus on mechanistic studies that can provide a firm underpinning for the underlying "functional selectivity hypothesis." The working hypothesis is that DHX and its N-n-propyl analog have full agonist actions at those D2-like receptors coupled to adenylate cyclase, whereas as these drugs are antagonists (or low efficacy partial agonists) at D2 receptors linked to potassium channels. Both DHX and N-p-DHX cause robust inhibition of adenylate cyclase in several models (e.g., inhibition of forskolin-stimulated or D1-mediated cCAMP synthesis/efflux in striatum, pituitary lactotrophs, or D2-transfected C-6 and MN9D clonal cells). Surprisingly, however, these same drugs have little or no effect on D2 receptors known (or presumed) to be coupled to potassium (K+) channels (e.g., they do not inhibit dopamine cell firing or dopamine release, and they induce only weak activation of K+ channels in pituitary lactotrophs). The development of drugs (such as the hexahydrobenzo[a]phenathridines) with functional selectivity may lead to dramatically improved pharmacotherapies by providing opportunities for targeting a subset of receptor-linked events, thus avoiding the undesirable side effects due to widespread activation or blockade of receptor functions. Such drugs would provide "pharmacological scalpels" for perturbing selected aspects. To begin to elucidate the mechanisms of functional selectivity, my experiments will focus on the actions of DHX on D2 receptor functions in rat striatum. The first aim is to collect concentration/response data to determine the potencies and efficacies of DHX and analogs at D2 receptors linked to adenylate cyclase or K+ channels. Superfused striatal slices will be used to compare effects on Da and ACh release (reflecting ion channel activation) with effects on cAMP efflux (an index of adenylate cyclase activation). Autoreceptor-mediated actions on adenylate-cyclase linked DA synthesis will be assessed by kinetic analysis of tyrosine hydroxylase. Aim 2 will extend results from in vitro studies to two in vivo paradigms, cerebral microdialysis (to measure DA, ACh and cAMP overflow) and the gamma-butyrolactone (GBL) model (to measure effects on DA synthesis). Experiments in Aim 3 will compare the pattern of G-protein activation by DHX and typical D2 agonists. Agonist-induced binding of [alpha-32P]GTP to G-proteins isoforms in striatal membranes and MN9D cells will be used as a marker of G-protein activation.

Biochemical Assay Core: A. SPECIFIC AIMS 1. Aim 1. Provide functional profiling using existing assays for compounds identified by the research projects or team researchers as being of potential interest. The primary goal of this core is to provide functional profiling of novel ligands and typical compounds. This includes some currently approved clinical drugs, interesting experimental ligands form the literature, and compounds identified from project research project, or suggested by external sources. There are currently in hand a series of biochemical functional assays that can distinguish typical agonists from functionally selective ligands. For an assay to be incorporated into this Core, it must be "routine," that is, a robust and reliable assay that has been validated by a project scientist or an external collaborator. Our previous research has shown that a battery of signaling assays that are independent or largely independent of each other will differentiate drugs with functionally selective properties, and allow selection of agents with novel properties. We believe that such differences in the pattern of signaling effects will not only differentiate the ligands from each other, but will also be predictive of preclinical behavioral and, possibly, clinical differences. Thus, this Core will feed select additional compounds into the two behavioral projects at Wyeth and at Duke. 2. Aim 2: Data analysis and assay improvement. In support of Aim 1, this Core shall also have several ancillary functions. First, it will work with Project scientists to look for new assays or cell lines (native or modified) that will enhance the sensitivity in detecting functionally selective ligands. Second, it will work to improve throughout (with sacrificing quantification) for assays that are heavily used. Third, we shall attempt to devise methods of data presentation that can be easily used by pharmacologists for studying functional selectivity.

drug discovery /isolation; laboratory mouse; stimulant /agonist

Project Summary/Abstract Dopamine D1 agonists have shown unique clinical potential for numerous disorders including cognitive deficits (e.g., from psychiatric or neurologic disorders or aging), attention deficit hyperactivity disorder, Parkinson?s disease, and cocaine abuse), and in the few reported clinical studies with experimental D1 agonists, the large effect sizes predicted from preclinical studies have been found. No brain available D1 agonist has yet been approved for clinical use largely because all high intrinsic activity D1 agonists were catechols, and had little oral bioavailability to justify clinical development. Pfizer Inc. has just reported non- catechol D1 agonists entering Phase III, and, although Phase II data are not public, their extensive studies in non-human primates (NHP) have confirmed the findings made with earlier, non-orally available compounds. This suggests that clinical use is on the horizon, raising numerous scientific questions, the most pressing of which is how the interactions of receptor occupancy and intrinsic activity translate into desired pharmacological effects. There are excellent D1 antagonist radioligands for in vivo imaging studies, but these ligands interact equivalently with active (high affinity G protein-coupled) and inactive (low affinity uncoupled) forms of the receptor. For this reason, they have been found to be insensitive to changes in dopamine signaling in vivo. We propose the discovery of a radiolabeled D1 selective agonist ligand that can be used in both laboratory and clinical imaging studies (e.g., SPECT) to quantify high affinity, functional receptors. We have selected a lead template based on excellent predicted pharmacodynamic properties and very accessible chemistry. We will make a precursor molecule that can be rapidly radioiodinated, and then deprotected to yield the candidate radioligand. We shall synthesize the intermediates and non-labeled predicted product, and validate the latter chemically and pharmacologically by its affinity and functional effects at dopamine receptors, and potential binding at off-target binding sites). Confirmation of its D1 agonist properties will be validated both behaviorally and physiologically, and we shall determine if it has metabolites that may affect its use. After the properties of the unlabeled probe have been confirmed, we shall develop and evaluate rapid and efficient radiosynthesis protocols for the candidate ligand. The proposed studies will use 125I, but are amendable to use of 123I (SPECT) or possibly PET (124I). We shall validate ligands by performing dose-response relationships, and compare the measured binding potential ex vivo with administered dose. We then shall select a tracer dose and perform competitive occupancy studies. Because we hypothesize that there are important in vivo differences between agonist and antagonist radioligands, we shall do parallel studies with the selective D1 antagonist [125I]- SCH28392, including autoradiography studies to compare the brain relative densities of high and low affinity sites. Finally, we shall validate the utility of the probe in vivo using functional assays for cognition and motor function.

PROJECT SUMMARY/ABSTRACT Agonists for dopamine D1?like receptors (D1R) consistently have been shown to enhance cognitive functions (e.g., working memory) in normal, lesioned, and aged murine and primate species, including humans. The D1R also plays important roles in the regulation of synaptic plasticity and cognitive process that are damaged during age-related cognitive decline such as in Alzheimer?s disease (AD). Receptor functional selectivity/biased signaling is a useful approach for discovery of drugs with ability to activate differentially signaling pathways mediated by a single receptor. Our recent work suggested D1 functional selectivity has critical influence in modulation of working memory related behavior and neurophysiology in the prefrontal cortex of young adult rats. These and other data suggested functionally selective D1 ligands could be promising candidates for age-related cognitive decline. Here we propose a discovery project to understand how signaling bias affects the cognitive effects of D1 ligands, and to target novel functionally selective D1 agonists that may become potential IND candidates for the pharmacological treatment of age-related cognitive decline. This will be accomplished by the following three iterative but also independent specific aims, using recent advances in three field, in vitro pharmacology, in vivo behavioral and physiological neuroscience, and in silico ligand-target mechanistic studies. Aim 1 will examine behavioral and neurophysiological changes manifested by differential activation of D1 signaling pathways in aged versus young rats (24-month elderly and 5-month young adult Fisher, TgF344 transgenic AD rats). Pre-selected D1 ligands will be tested by a working memory related delayed alteration response task in the T-maze primarily, for their effects on behavior and neurophysiology. Aim 2 will use computational methods for ligand-target simulation to develop mechanism and accelerate drug discovery. Aim 3 will complete a thoroughly pharmacological screen to elucidate optimal D1 signaling profiles for age-related cognitive decline. This will involve characterization of receptor binding properties and functional assays, and off- target analysis, in not only heterologous expression systems but also brain tissue. The successful completion of proposed specific aims will provide heuristic information on the potential advantages of functionally selective D1 agonists for age-related cognitive decline. Recent clinical studies have shown, contrary to earlier views, that the D1R is a druggable target. Thus, this project will provide a rational foundation for selection of novel candidates for future IND-enabling studies and clinical trials.