Remi Martin-Fardon - US grants

DESCRIPTION (provided by applicant): Neural circuits implicated in drug conditioning, craving, and relapse overlap with those involved in natural reward. Recently, the orexin/hypocretin (Orx/Hcrt) system has been identified to regulate a range of physiological processes, including feeding, energy metabolism, and arousal, and has been shown to be recruited by drugs of abuse. Orx/Hcrt neurons are predominantly located in the lateral hypothalamus (LH), and accumulating evidence indicates an important role for these neurons in drug addiction. These Orx/Hcrt neurons project to the paraventricular nucleus of the thalamus (PVT), a region that has been identified as a way- station that receives projections from the LH, processes information, and then modulates the mesolimbic and extrahypothalamic stress systems. While not thought to be part of the cocaine-seeking circuitry, evidence implicates the PVT in the modulation of reward function in general and drug-directed behavior in particular. Importantly, a correlation between cocaine-seeking behavior and activation of the PVT has been detected, but not in the case of natural reward-seeking behavior. This suggests that cocaine dysregulates the neurotransmission within the PVT. Capitalizing on our findings, we hypothesize that following repeated cocaine use, the Orx/Hcrt system acquires a preferential role in mediating drug of abuse seeking vs. natural reward seeking. This proposal is designed to study the neurobiological basis of chronic vulnerability to relapse by focusing on Orx/Hcrt transmission in the PVT as a novel neural substrate that may be responsible for the distinctly compulsive nature of cocaine seeking as opposed to behavior motivated by natural rewards essential for survival, well being, and healthy hedonic pursuits. Specifically, this proposal will (i) behaviorally characterize the specific implication of the PVT in cocaine seeking, (ii) investigate cocaine-induced neuroplastic changes within Orx/Hcrt transmission and (iii) investigate the effects of dysregulated Orx/Hcrt-PVT transmission on the mesolimbic system.

DESCRIPTION (provided by applicant): The development of alcohol use disorders follows a transition from social use motivated by hedonic and anxiolytic effects to dependence motivated by increasing withdrawal symptoms and an evolving desire to drink during abstinence. In this latter stage of alcohol dependence, abstinence from drinking is often accompanied by negative emotional symptoms, such as increased anxiety and depression, and the alleviation of these negative emotional states is hypothesized to be a major driving force for continued alcohol consumption. This shift from positive to negative reinforcement mechanisms likely results from enduring changes in CNS function induced by excessive alcohol consumption. Although several signaling systems have been implicated in this process there is still an incomplete understanding of the neural mechanisms underlying alcohol dependence. We have gathered evidence that EtOH consumption increases levels of the endogenous cannabinoid (eCB) 2- arachidonoyl glycerol (2-AG) in rodent brain, while long-term intermittent EtOH exposure down-regulates eCB signaling in brain regions relevant to emotional processing. Dependence-associated anxiety-like behavior and excessive EtOH consumption are reduced by generalized enhancement of eCB tone, though similar manipulations do not produce these effects in non-dependent animals. Based on these findings, we hypothesize that eCB clearance inhibitors have therapeutic value for treating alcohol dependence and alcoholism. This hypothesis will be tested through three Specific Aims. Aim 1 will characterize the ability of highly selective eCB clearance inhibitors to alleviate anxiety-like behavior in EtOH dependent mice throughout a period of protracted withdrawal. Importantly, these experiments will characterize the relative influence of two primary eCB molecules, 2-AG and anandamide (AEA), by selectively inhibiting the distinct hydrolytic mechanisms that clear these lipids from the brain. The experiments in Aim 2 will employ biochemical and neurochemical approaches to characterize the mechanisms contributing to dependence-associated dysregulation of brain eCB signaling. Additional work in this Aim will evaluate the influence of eCB dysregulation on other neurotransmitter systems involved in withdrawal-associated anxiety-like behavior and excessive EtOH consumption (including glutamate, serotonin and norepinephrine). The experiments in Aim 3 will characterize the efficacy of selective eCB clearance inhibitors for reducing high levels of EtOH consumption associated with dependence and protracted withdrawal. These experiments will also characterize the influence of eCB signaling on binge-like ethanol intake in non-dependent mice. Completion of the proposed work is likely to highlight a previously unrecognized mechanism in the etiology of alcohol dependence and may identify novel therapeutic targets for alcoholism.

DESCRIPTION (provided by applicant): Impaired cognitive processing is a hallmark of addiction. Deficits in inhibitory control (impulsive action), poor evaluation of consequences (impulsive choice) and ineffective reversal of compulsive or habitual behaviors (cognitive flexibility) can propel continued drug use despite adverse consequences. A persistent question in alcohol research regards the relative influence of pre-existing cognitive disruptions that confe susceptibility to problem drinking versus the induction of cognitive impairment related to cortical damage induced by repeated alcohol intoxication and withdrawal. In this regard animal models can provide important insight into the etiology of alcohol-induced cognitive impairment and can provide a platform for mechanistic studies and rapid pharmacotherapy screening. Using behavioral paradigms analogous to clinically employed tasks we have gathered preliminary evidence of significant increases in impulsive action and impulsive choice behaviors that emerge in rats during protracted withdrawal from long-term intermittent alcohol consumption. These cognitive impairments persist for several weeks, and can be ameliorated by a pharmacological manipulation known to improve cognitive function in human alcoholics. Based on these findings and knowledge of the neural mechanisms governing these behaviors in rats we hypothesize that withdrawal-associated dysregulation of monoamine and amino acid signaling in frontal cortical regions contributes to increased impulsivity and deficient cognitive flexibility during protracted alcohol withdrawal. This hypothesis will be tested through three Specific Aims. Aim 1 will characterize the emergence, nature and persistence of cognitive disruption during protracted alcohol withdrawal. Different facets of impulsive action will be explored using a novel 5-Choice Continuous Performance Task (5C-CPT) and the Stop Signal Reaction Time task (SSRT). Impulsive choice behavior will be indexed using the Delay Discount Test, and cognitive flexibility will be assessed using an operant spatial reversal learning task. The experiments in Aim 2 will employ in vivo microdialysis and biochemical approaches to characterize monoamine, and amino acid function in the orbitofrontal and medial prefrontal cortices during protracted alcohol withdrawal. Aim 3 will evaluate the efficacy of pharmacologic agents for ameliorating withdrawal-associated increases in impulsive action and impulsive choice.

? DESCRIPTION (provided by applicant): The hypocretin (Hcrt) system has long been known to regulate a wide range of physiological processes, including feeding, energy metabolism, and arousal. More recently, concordant observations have demonstrated an important role for Hcrt in the reinforcing properties of most drugs of abuse. Accordingly, Hcrt neurons, which predominantly arise from the lateral hypothalamus (LH), project to brain structures implicated in the regulation of arousal, stress, and reward. Although Hcrt neurons have been shown to massively project to the paraventricular nucleus of the thalamus (PVT), recent evidence suggests that the PVT may be a key relay of Hcrt-coded reward-related communication between the LH and both the ventral and dorsal striatum. While this thalamic region was not thought to be part of drug addiction circuitry, an increasing amount of evidence indicates that the PVT-particularly Hcrt transmission in the PVT-is implicated in the modulation of reward function in general and several aspects of drug-directed behaviors in particular. Importantly, findings from our laboratory demonstrated selective activation of the PVT during ethanol seeking, and our preliminary data suggest that a history of ethanol dependence produces a downregulation of Hcrt in the LH and upregulation of Hcrtr1 (encoding Hcrt receptor 1) in the PVT. This proposal is designed to study the neurobiological basis of chronic vulnerability to relapse by focusing on Hcrt transmission in the PVT as a novel neural substrate that may be responsible for the compulsive nature of ethanol seeking. Specifically, this proposal will (i) establish the role of Hcrt transmission in the PVT in ethanol-seeking behavior and (ii) test the hypothesis that knocking down Hcrt using local gene silencing in the LH in nondependent rats will mimic the phenotype of postdependent rats. Overall, the planned experiments will provide novel insights into the specific involvement of LH?PVT Hcrt transmission in alcohol-seeking behavior and will likely highlight a previously unrecognized neurotransmission system in the etiology of compulsive alcohol seeking during abstinence and justify targeting the hyprocretin system for alcoholism and relapse prevention.

Endogenous cannabinoid (eCB) signaling in the brain plays a homeostatic role in the constraint and termination of stress responses. During the previous cycle of the TSRI-ARC we found that chronic intermittent EtOH exposure down-regulates eCB signaling in the central nucleus of the amygdala, a brain region critically involved in stress responses and emotional processing. We also found that dependenceassociated anxiety-like behavior and excessive EtOH consumption are alleviated by enhancement of eCB tone. Endocannabinoids are also present in other stress-responsive brain regions such as the basolateral amygdala (BLA) and bed nucleus of the stria terminalis (BNST) where they play a prominent role in the plasticity of excitatory and inhibitory signaling. Dysregulated synaptic function in these regions is believed to contribute to dependence-associated affective disorders and we have gathered preliminary evidence that eCB clearance mechanisms are disrupted in these regions by alcohol dependence. Based on these observations we hypothesize that dysregulated eCB signaling in the BLA and BNST contributes to affective dysregulation and excessive EtOH consumption associated with long-term EtOH exposure. This hypothesis will be tested through three Specific Aims. Aim 1 will employ biochemical and neurochemical approaches to characterize the nature and persistence of dysregulated eCB function resulting from excessive EtOH exposure. Aim 2 will characterize the influence of disrupted eCB function in the BLA and BNST on dependence-associated anxiety-like behavior over a period of protracted abstinence. The relative influence of two primary eCB molecules, 2-AG and anandamide (AEA) will be characterized using pharmacological and genetic manipulations of their respective clearance mechanisms. Aim 3 will characterize the efficacy of selective eCB clearance inhibitors for reducing high levels of EtOH consumption associated with dependence and protracted withdrawal. The experimental design incorporates two distinct animal models of excessive drinking to index the development of eCB disruptions along the trajectory from chronic binge drinking to excessive EtOH consumption motivated by dependence.

Abstract    A central problem in the treatment of ethanol (EtOH) addiction is the prevalence of relapse to EtOH use even  after  protracted  intervals  of  forced  or  self-­imposed  abstinence.  Advances  have  been  made  in  elucidating  the  neurocircuitry that mediates craving and EtOH seeking, which provides insights into the neurobiological basis  of  relapse.  Functional  brain  imaging  in  humans  and  studies  that  use  c-­fos  expression  as  a  marker  of  neural  activation  in  rodents  implicate  interconnected  cortical  and  limbic  brain  regions  in  response  to  drug  cue-­,  drug  priming-­,  and  stress-­induced  reinstatement.  Major  components  of  this  circuitry  include  the  medial  prefrontal  cortex (mPFC), basolateral amygdala (BLA), central nucleus of the amygdala (CeA), bed nucleus of the stria  terminalis (BNST), ventral tegmental area (VTA), nucleus accumbens (NAC), hippocampus, thalamus (THAL),  and dorsal striatum. The hypocretin (Hcrt) system regulates a wide range of physiological processes, including  feeding, energy metabolism, arousal, and stress, and is recruited by drugs of abuse. Of interest for the present  proposal,  recent  studies  have  demonstrated  a  critical  contribution  of  Hcrt  in  the  modulation  of  stress  and  a  possible anxiolytic effect of Hcrt receptor (Hcrt-­r) antagonists. Furthermore, we have collected convincing data  that  EtOH  exposure  recruits  the  Hcrt  system.  Specifically,  we  found  that  downregulation  of  Hcrt  mRNA  was  observed  in  the  lateral  hypothalamus  (the  major  source  of  Hcrt  production)  of  animals  that  had  a  history  of  EtOH dependence and that blockade of Hcrt-­r selectively reversed EtOH seeking vs. natural reward seeking.  Chronic  drug  use  is  well  known  to  dysregulate  stress  responses  that  are  mediated  by  corticotropin-­releasing  factor  (CRF)  in  both  the  hypothalamic-­pituitary-­adrenal  (HPA)  axis  and  extrahypothalamic  brain  stress  areas  outside  the  HPA  axis  (e.g.,  CeA  and  BNST).  With  repeated  cycles  of  drug  use,  the  HPA  axis  becomes  hyporesponsive,  accompanied  by  an  increase  in  the  extrahypothalamic  CRF  stress  system  response  (i.e.,  CRF-­CRF1  receptors).  Importantly,  a  Hcrt/CRF  interaction  exists,  and  it  has  been  proposed  that  Hcrt  modulation  of  CRF  neurons  participates  in  the  chronic  relapsing,  negative  affective  states  that  characterize  drug addiction. Converging lines of evidence from human and animal studies suggest that impairment of mPFC  function due to drugs of abuse exposure is a key factor in the transition from goal-­directed to compulsive drug  seeking. The mPFC contains CRF interneurons, and Hcrt neurons project to the mPFC. This proposal will test  the hypothesis that a history of EtOH dependence dysregulates Hcrt and its interaction with CRF in the mPFC,  particularly  the  infralimbic  area  (IL),  and  if  this  dysfunction  will  predict  compulsive  EtOH  seeking  (relapse)  precipitated by stress during acute/early, late, and protracted abstinence that could explain compulsive EtOH  seeking. From the perspective of future medication development, this project is likely to highlight a previously  unrecognized mechanism in the etiology of compulsive EtOH seeking during abstinence;? ultimately leading to  the identification of novel therapeutic targets for the prevention of EtOH relapse.    

A central problem in the treatment of ethanol (EtOH) addiction is the prevalence of relapse to EtOH use even after protracted intervals of forced or self-imposed abstinence. Advances have been made in elucidating the neurocircuitry that mediates craving and EtOH seeking, which provides insights into the neurobiological basis of relapse. Functional brain imaging in humans and studies that use c-fos expression as a marker of neural activation in rodents implicate interconnected cortical and limbic brain regions in response to drug cue-, drug priming-, and stress-induced reinstatement. Major components of this circuitry include the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST), ventral tegmental area (VTA), nucleus accumbens (NAC), hippocampus, thalamus (THAL), and dorsal striatum. The hypocretin (Hcrt) system regulates a wide range of physiological processes, including feeding, energy metabolism, and arousal, and is recruited by drugs of abuse, including EtOH. Of interest for the present proposal, recent studies have demonstrated a critical role of Hcrt in the modulation of stress and a possible anxiolytic effect of Hcrt receptor (Hcrt-r) antagonism. Hcrt neurons, located only in the hypothalamus, project to the major components of the neurocircuitry that mediates EtOH seeking and innervates densely the paraventricular nucleus of the thalamus (PVT). Recent evidence suggests that the PVT participates in the modulation of reward function in general and drug-directed behavior in particular. Furthermore, it has been shown that PVT is an important contributor in the regulation of stress, a critical factor that can induce intense craving and trigger relapse in abstinent individuals. Earlier findings demonstrated selective recruitment of the LH/DMH/PFA-PVT during EtOH seeking, and preliminary data suggest that a history of EtOH dependence dysregulates the Hcrt/Hcrt-r system. Thalamic impairments are a key feature of EtOH-related brain dysfunction in alcoholics, remaining to be determined are the extent to which a history of EtOH dependence dysregulates Hcrt and whether this dysfunction will predict maladaptive compulsive EtOH-seeking (relapse) that is precipitated by stress vs. normal (food-seeking) behavior. This proposal will test the hypothesis that a history of EtOH dependence dysregulates Hcrt and its interaction with the PVT, and that this dysfunction will predict compulsive EtOH seeking (relapse) precipitated by stress. Furthermore, using local gene silencing, this proposal will test the hypothesis that the permanent decrease in Hcrt production via a viral vector will prevent Hcrt transmission dysregulation in the PVT during dependence and therefore prevent exacerbated response to stress during EtOH abstinence. This project is likely to highlight a previously unrecognized mechanism in the etiology of compulsive EtOH seeking during abstinence; ultimately leading to the identification of novel therapeutic targets for the prevention of EtOH relapse.

PROJECT SUMMARY Alcohol use disorder (AUD) is a serious condition with severe medical and societal consequences.There has been little progress in medical treatment over the past decades. We are taking a translational approach and have established an animal model, where alcohol-dependent rats in various stages of abstinence are subjects of investigation. The neuronal target is the opioid systems and the opponent hypothesis. We propose that the initial euphoric effects are channeled through the enkephalin/mu-opioid receptor (MOP) and the later developing negative reinforcement (craving) is related to activity at the dynorphin/kappa-opioid receptor (KOP). In fact, the MOP antagonist, naltrexone is a FDA-approved agent with indication to reduce relapse. KOP antagonists are entering clinical trials in different neuropsychiatric conditions. We have chosen CERC-501 as index drug, being reversible and apparently well tolerated in clinical examination. After behavioral recordings, brain specimens will be dissected and form a ?brain bank? for further analysis. A focus of interest is the central nucleus of the amygdala (CeA), and the circuitry presenting MOP and/or KOP. Selected specimens will undergo superresolution microscopy (quantitative Single Molecule Localization Microscopy, qSMLM). A pilot study showed that already an acute dose of EtOH disrupts localization of MOP and KOP, an effect blocked by naltrexone. How EtOH affects receptor mobility in the plasma membrane, receptor clustering (homo- and hetero-dimers) and association with protein- and lipid-rich membrane domains will be studied by fluorescence correlation spectroscopy (FCS). As with qSMLM resolution is achieved at the single-molecule level. Both technologies will be used to study co- localization of receptors with proteins of relevance for the signaling cascade. We hypothesize that EtOH perturbs the dynamic selforganization of signaling domains harboring MOPs and KOPs, that distinct alterations in mechanisms controlling MOP vs KOP organization develop during chronic EtOH exposure. With OP antagonists innate MOP and KOP signaling complexes are stabilized at the nanoscale level. Focused studies of these mechanisms will provide critical new insight into molecular mechanisms through which EtOH-induced receptor disruptions may be prevented or reversed. The commensurate importance of the two opioid systems may vary between individuals and influence the choice of personalized therapy with OP antagonists. Studies will include the N40D MOP genotype known to affect the behavioral phenotype and sensitivity to naltrexone.