Jay P. McLaughlin - US grants

DESCRIPTION (provided by applicant): Released endogenous opioid peptides regulate pain sensation, physiological stress responses and reward pathways. As such, endogenous opioids are believed to act as key modulators of the behavioral response to stress as well as drug self-administration. This proposal will assess the role of the endogenous kappa opioid system in the mediation of behavioral responses to stress and drug reward. In this proposal, we will perform a series of behavioral experiments exposing mice to the forced swim test, inducing a stress response mediated by the release of endogenous dynorphin peptides. Pilot results with C57BI/6 mice show that the mild stress induced by the forced swim test produced increases in immobility and tail-flick latency which were blocked by preadministration of the kappa opioid antagonist nor-BNl. Moreover, the increases in swimming immobility and tail-flick latency were observed in wild-type mice exposed to the forced swim stressor, but not in littermates lacking Dynorphin gene products. Future studies are planned to characterize the endogenous opioid peptides that are released in response to the swim stressor, as well as a parametric analysis to fully characterize the component of the stressor mediated by the endogenous kappa opioid system. In addition, these studies will be extended to examine the role of stress-induced endogenous kappa opioid activity in the response to rewarding drugs. Preliminary evidence shows that prior exposure of mice to a forced-swim stress induces a potentiation of the conditioned place-preference response to cocaine. In contrast, mice pretreated with nor-BNI or lacking dynorphin gene products prior to forced swimming did not show a stress-induced potentiation of cocaine conditioned place preference. Future studies with dynorphin wild type and knockout mice are planned wherein the duration of the stress effect and a parametric analysis of forced-swim stress impact on drug reward are examined by measuring the conditioned place preference for cocaine and heroin. In summary, it is expected that this data set would characterize a response to behavioral stress and drug reward regulated by the action of the endogenous kappa opioid system. A better understanding of the relationship between stress and endogenous kappa systems may lead to new insights into stress-induced relapse of drug abuse, as well as provide a new approach for therapeutic intervention in these phenomena.

DESCRIPTION (provided by applicant): Endogenous opioid peptides are believed to modulate the physiological response to stress, regulating pain sensation, reward pathways and behavioral responses to the environment. This proposal will assess the mediating role of the endogenous kappa opioid system in responses to a mild stressor, the forced swim test. Mice will be exposed to forced swimming to induce the release of endogenous opioid peptides known to activate kappa opioid receptors. Initial results show that the mild stress induced by repeated forced swim testing increased behavioral immobility and subsequent tail flick latency through a mechanism sensitive to the kappa opioid antagonist, nor-BNI or dynorphin gene disruption. Further assessment of the endogenous activation of kappa opioid receptors is possible with a novel antibody probe (KOR-P) that can distinguish the agonist-activated form of the receptor. When opioid receptors are activated by applied agonist or endogenously released opioid peptide, G-protein receptor kinase phosphorylates the serine-369 residue on the kappa opioid receptor to initiate the desensitization process. Preliminary data suggest that the KOR-P antibody can distinguish the phosphorylated kappa opioid receptor from the basal state, and thus may be used as a probe in Western blot and immunocytochemical analyses to detect prior activation of the kappa opioid system. Pilot Western blot results demonstrate that the forced-swim stress induced an increase in KOR-P antibody labeling of brain protein isolated from tested mice. The increase in specific labeling was blocked by administration of nor-BNI prior to swim testing. Future studies are planned to determine the endogenous opioid peptides that are producing the kappa opioid receptor activation in response to the swim stressor, starting with examination of dynorphin knockout mice and their wild-type littermates in the forced swim test. In addition, studies will be extended to refine immunocytochemical methods and determine where in the brain kappa opioid receptor activation occurs in response to environmental stress. It is expected that this data set would define a functional neural circuit activated by behavioral stress and regulated by the action of the endogenous kappa opioid system. A better understanding of the relationship between stress and endogenous kappa systems may lead to new insights into such disorders as depression and relapse of drug abuse, possibly providing new therapeutic approaches in these syndromes.

DESCRIPTION (provided by applicant): Depression and anxiety are prevalent mood disorders symptomatic of the syndrome described as NeuroAIDS, and pose significant problems for the treatment and well-being of HIV-positive patients. Unfortunately, the biological mechanisms linking HIV-related neuropathology to the dysfunction of neuronal circuitry and the progression of behavioral mood disorders are not well understood. Recent evidence suggests that HIV-accessory proteins such as Tat may spread throughout the brain from HIV-infected cells, producing neurotoxicity and changes in neuronal activity that could account for the behavioral changes. We hypothesize that Tat protein expression is sufficient to produce neurodegeneration and dysfunction of neuronal circuitry mediating mood, resulting in an increase of depression- and anxiety-like behaviors. This proposal utilizes the GT-tg transgenic mouse and its doxycycline-gene induction strategy for a controlled, selective expression of Tat protein in the brain. Induced GT-tg bigenic mice will be used to test the hypothesis with behavioral and imaging studies that Tat-mediated increases in depression- and anxiety-like behaviors are correlated with Tat-induced alterations in brain structure and function in regions associated with depression and anxiety, including the amygdala, anterior cingulate, orbitofrontal cortex and hippocampus. Behavioral studies with these mice will assess how Tat expression affects depression-like behaviors with the forced swim stress and social aversion tests. Behavioral experiments also will examine effects of Tat expression on anxiety-like behaviors using the open field, elevated plus maze, light-dark box, acoustic startle and marble burying tests. Assays of Tat mRNA and protein levels will correlate expression of Tat to the observed changes in behavior. In preliminary studies, Tat-induced mice spent less time than uninduced littermates in social interactions and more time immobile in forced swim stress tests, suggestive of a Tat-mediated increase in depression-like behavior. Tat-induced mice also demonstrated increased anxiety-related behaviors, with less time spent in open-field environments and a 3-fold increase in marble burying. Concurrently, we will examine the effects of Tat protein on brain structure and cell death using ex vivo magnetic resonance imaging (MRI) at ultra high magnetic field strength and TUNEL staining for apoptosis in Tat-induced animals. Preliminary ex vivo structural imaging studies documented significant reductions in the grey matter density of amygdala and hippocampus in Tat-expressing mice. Additionally, we will use BOLD functional MRI with corticosterone challenge to identify functional changes in brain regions associated with mood disorders resulting from varying levels of induction and duration of exposure to Tat protein and the response to a challenge dose of corticosterone. Overall, this project seeks to prove that functional and structural deficits can be detected in the brain circuitry of Tat-induced animals, thereby defining mechanisms by which Tat may mediate the mood disorders characteristic of NeuroAIDS.

? DESCRIPTION (provided by applicant): Substance abuse and HIV-1 infection are highly comorbid, and HIV-1 infection correlates with the progression of neuropsychological dysfunctions identified as NeuroAIDS. However, relatively little is known about HIV modulation of the rewarding effects of abused drugs. Biological mechanisms linking HIV-related neuropathology to neural dysfunction and the progression of behavioral abnormalities in vivo are not well understood. Recent epidemiological evidence suggests that the HIV regulatory protein Tat and other HIV-proteins may spread throughout the brain from HIV-infected cells, inducing neuronal dysfunction that could account for behavioral changes. We hypothesize that exposure to Tat exerts neurochemical changes in forebrain circuitry mediating reward, thereby potentiating morphine's rewarding effects. This proposal utilizes the GT-tg transgenic mouse and its conditional gene induction strategy via doxycycline (Dox) administration, which enables controlled, brain-selective expression of Tat protein, to assess Tat effects on opioid-related effects and behaviors. Dox-induced GT-tg bigenic mice will be studied to assess whether Tat potentiates morphine conditioned-place preference (CPP) and voluntary morphine consumption (using a two-bottle choice (TBC) assay) compared to mice lacking Tat. Additional studies will evaluate Tat-induced reinstatement of extinguished morphine-seeking behavior. We will also use magnetic resonance spectroscopy (MRS) to determine whether Tat protein alters medial frontal cortex glutamate and N-acetylaspartate levels, which could reflect altered neurotransmission and neurotoxicity. Lastly, we will use behavioral and MRS methodology to assess whether the anti-inflammatory agent indomethacin, the NMDA-receptor (NR2B-selective) antagonist ifenprodil, or the small molecule Tat protein inhibitor didehydro-Cortistatin (dCA) can inhibit or reverse Tat-mediated effects. Tat protein expression in postmortem brain samples after treatment and testing will be confirmed by ELISA and Western blot analysis. Our pilot behavioral data suggest that Tat both potentiates morphine-CPP and induces reinstatement of extinguished morphine-seeking behavior. Pilot MRS studies of medial frontal cortex found decreases in glutamate and N-acetylaspartate in Dox-treated GT-tg mice versus baseline measures in these mice. Pilot data also suggest that prophylactic pretreatment with indomethacin and dCA prevented Tat-induced potentiation of morphine CPP. Overall, this project is designed to extend pilot findings, and lead to a better understanding of how Tat contributes to the neuropathology produced during HIV infection, including the possible modulation of behavioral motivation for opioids. Study findings may also identify novel mechanisms that could be exploited as therapeutic interventions for development to treat comorbid HIV/opioid abuse.

Perturbation of dopaminergic transmission is implicated as a risk factor of HIV-1 associated neurocognitive disorders (HAND). Dopaminergic system plays a causal role in drug rewarding and modulation of the brain function including cognition. Prefrontal cortex is an important brain region for higher cognitive function, where norepinephrine transporter (NET) is the primary mechanism of homeostatic regulation of stable synaptic dopaminergic tone. HIV-1 Tat protein and cocaine synergistically increase synaptic dopamine levels, thereby producing neurocognitive impairment. Our initial findings show that in vitro exposure to recombinant Tat1-86 inhibits dopamine and norepinephrine reuptake by dopamine transporter (DAT) and NET and Tat binds to DAT and NET through a direct protein-protein interaction. We have demonstrated that Tat-induced inhibition of DAT function is mediated by binding to allosteric binding site(s) on DAT, not by interacting with the DA uptake site. Accordingly, attenuating Tat binding to DAT would be expected to have minimal influence on physiological DA transport. Indeed, our recent findings show that a novel quinazoline series of allosteric modulators decrease cocaine potency for inhibition of DA uptake and attenuate Tat-induced inhibition of DA reuptake and cocaine binding by DAT. We hypothesize that Tat, via the unique allosteric modulatory sites, perturbs the DAT and NET regulatory network that normally sustains concentrative DA or NE transport and potentiates cocaine?s effect on DAT and NET, resulting in DA/NE-linked neuropsychiatric dysfunction prominently featured in HAND. We will (Aim 1) Identify the binding sites for Tat in human NET, and explore allosteric modulation of this transporter by Tat and cocaine; (Aim 2) determine the pathogenic role of DAT/NET-mediated dopaminergic transmission in inducible Tat transgenic mice by assessment of Fast-scan cyclic voltammetry and whole cell patch clamp recording; and (Aim 3) perform proof of concept studies using novel allosteric modulators to establish their potential for therapeutic application in HAND using integrated computational modeling, pharmacological, and behavioral approaches. Our long-term goal is to explore new ways to target DAT/NET for therapeutic interventions to improve neurocognitive dysfunction of HAND in concurrent cocaine abusers.