Jocelyn M. Richard, PhD - US grants

PROJECT SUMMARY Aberrant motivation is an important feature of a variety of human mental disorders. Excessive appetitive motivation is implicated in drug addiction and obesity, leading to sometimes compulsive pursuit and overconsumption of drugs or food (1, 2). The nucleus accumbens (NAc), embedded within mesocorticolimbic circuits, plays an important role in excessive incentive motivation. The medial shell region of the NAc is particularly associated with the generation of appetitively motivated behaviors. Beyond the well known role in incentive motivation, mesocorticolimbic systems may also mediate some fearful states. This proposal focuses on the role of local glutamate disruptions in rostral NAc shell in producing positively-motivated behaviors like intense feeding, and the role of glutamate disruptions in caudal zones of NAc shell in producing negatively- motivated behaviors. Mesolimbic dopamine (DA) systems interact with corticolimbic glutamate disruptions for appetitive and fearful motivational salience (5): Aim 1 will identify whether D1 or D2 receptor subtypes are necessary for generation of either positively-motivated behaviors (feeding) in rostral shell or negatively- motivated behaviors (defensive treading) following AMPA receptor blockade. It is also important to understand the larger corticolimbic circuit in which NAc shell is embedded, and particularly to understand how corticolimbic top-down signals regulate generation of appetitive and defensive motivation by NAc. Aim 2 will identify which region of PFC is primarily responsible for modulation of subcortical unconditioned positive and negative motivation: medial OFC, infralimbic or prelimbic. Finally, it is important to understand how psychological context modulates NAc generation of fear and feeding by recruiting corticolimbic circuits. This phenomenon may be relevant to contextual influences on human pathological motivations, as well as to therapeutic interventions. Aim 3 will identify the particular PFC inputs to NAc that mediate environmental modulation of NAc-generation of motivation.

DESCRIPTION (provided by applicant): A large body of evidence points to the importance of endogenous opioids in alcohol drinking and reinforcement (1). The medial shell of nucleus accumbens (NAc) has emerged as an important neural substrate for these effects and may mediate problem alcohol use (2-9). Importantly, infusion of the mu-opioid receptor agonist, DAMGO, into rodent NAc potentiates alcohol consumption (10), yet the behavioral and neurological mechanisms of this effect remain unknown. Medial NAc shell is a critical relay for prefrontal cortical influences on both hypothalamus and ventral pallidum, forming an extinction circuit which may co -opt mechanisms normally devoted to satiety, and modulation of this circuit may serve as a critical mechanism of opioid-induced alcohol consumption and relapse (11-12). Glutamatergic inputs from infralimbic cortex to medial NAc shell are posited to suppress drug and alcohol seeking following extinction and during reinstatement (13- 14), potentially via inhibitory projections from NAc to ventral pallidum and lateral hypothalamus (11, 15). The proposed experiments will systemically test the psychological and neural mechanisms of NAc DAMGO- induced alcohol consumption, and in particular the effects of NAc shell DAMGO on this extinction circuit. The experiments in Aim 1 of this proposal will test the specific effects o NAc shell DAMGO in alcohol self - administration and cue-induced relapse to alcohol seeking, to determine what aspects of alcohol reward are enhanced by NAc mu-opioid signaling (i.e. palatability, motivation, or reinforcement). It is critical to understand how NAc shell mu-opioid signaling affects top-down inputs from infralimbic cortex that may normally suppress alcohol seeking and relapse. Aim 2 of the research plan will test whether selective activation of infralimbic inputs to NAc can suppress alcohol consumption and relapse, and whether NAc shell DAMGO blocks these effects, during alcohol self-administration and relapse. It is also vital to understand how NAc mu-opioid induced changes in alcohol seeking are encoded in downstream structures of the extinction circuit, including ventral pallidum and lateral hypothalamus, which may encode different aspects of alcohol reward. The experiments in Aim 3 will test whether neural activity in lateral hypothalamus or ventral pallidum encodes alcohol seeking, consumption, or relapse to alcohol seeking, or enhancements in any of these processes following NAc shell DAMGO.

Project Summary Acute or chronic stress contributes to both escalation of alcohol use and relapse to alcohol seeking after abstinence. Stressors are hypothesized to contribute to relapse in part by promoting responses to cues in the environment that have been previously associated with alcohol availability, yet the brain mechanisms of this interaction between cues and stress are poorly understood. An area of the brain implicated in both stress- and cue-induced reward seeking is the ventral pallidum (VP). I have previously found that VP neurons encode the vigor of cue-elicited reward seeking, and my preliminary results suggest that VP neurons encode reinstatement of reward seeking by the pharmacological stressor, yohimbine. In the current proposal, I aim to confirm and expand my findings regarding VP encoding of reinstatement by stress, using a behavioral stressor, and to further dissect the neural circuit mechanisms by which VP neurons contribute to alcohol seeking. Here, in the K99 phase, I will examine VP encoding of cued alcohol seeking and reinstatement induced by stress in dependent versus non-dependent alcohol seeking rats. This aim will provide me with new training in models of alcohol dependence and stress reinstatement. I will then examine encoding of alcohol seeking behavior in inputs to VP from the prefrontal cortex (PFC) and basolateral amygdala (BLA), using a novel method for projection specific measurement of activity. This method, fiber photometry, uses viral-based expression of a fluorescent calcium indicator paired with optical measurement of fluorescence. Training in this skill will allow me to measure projection-specific neural activity in the proposed aims and future projects. Further, this aim will provide critical information regarding the timing of activity in BLA and PFC inputs during alcohol seeking, to be tested in the R00 phase. I will also assess the collateralization of these neurons, comparing two methods: traditional sectioning and immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). This method provides unprecedented access to the three-dimensional structure of immunolabeled cells, and the opportunity to obtain both large-scale and high resolution information simultaneously, crucial for planned experiments in my future laboratory. In the R00 phase I will test the functional role of temporally precise activity in BLA and PFC inputs to VP during cued alcohol seeking and reinstatement of alcohol seeking, using optogenetics. I will also measure activity in genetically-defined populations of neurons projecting from the nucleus accumbens (NAc), which include both D1- and D2-dopamine receptor dominant neurons. Using transgenic rats I will selectively express calcium indicators in either D1- or D2-dominant neurons projecting to VP. These experiments will provide novel data regarding the differential activity of D1 and D2 neurons projecting to VP and will provide an important framework for future experiments and grants. These experiments will characterize the neural circuit mechanisms by which VP neurons drive alcohol seeking, and how they differ across different states of dependence and following stress.

Project Summary A hallmark of alcohol use disorder is continued seeking and consumption of alcohol despite negative consequences. Neuroadaptations in corticostriatal projections to nucleus accumbens are critical for the development of compulsive-like alcohol use behaviors, including in an aversion-resistant drinking model. Yet it remains unclear how potentiated activity in nucleus accumbens neurons alters sensitivity to aversive outcomes during consumption and seeking of alcohol. Pre-existing individual differences in aversion-related circuits have been shown to predict future compulsive-like alcohol consumption. Yet, we are not aware of any demonstrations of alcohol-induced neuroadaptations in aversion-related circuits that track the development of aversion-resistant drinking, and that account for the emergence of this compulsive phenotype after alcohol exposure. Our long-term goal is to identify dynamics changes in the activity of aversion-related neural circuits that drive the emergence of compulsive alcohol use. Here, we will examine glutamatergic basal forebrain neurons that project to the lateral habenula. These neurons are found in an anterior-posterior continuum from the ventral pallidum to the lateral hypothalamus and are targeted by nucleus accumbens inhibitory projection neurons. We hypothesize that the emergence of aversion-resistant drinking requires selective inhibition of these neurons during alcohol consumption. A crucial first step in our investigation of this circuit is to determine whether the emergence of aversion- resistant drinking is correlated with cell-type specific alterations in drinking-related neural activity in the glutamatergic basal forebrain. We will assess this question, and whether these neural correlates are downstream of nucleus accumbens mechanisms in Aim 1. Our second goal is to examine lateral habenula glutamate activity dynamics during aversion-resistant drinking. Therefore, our Aim 2 experiments will utilize a fluorescent glutamate sensor combined with fiber photometry to identify temporally-specific neural correlates of aversion-resistant drinking. Finally, in Aim 3 we will use chemogenetic approaches to assess the functional contributions of basal forebrain projections to lateral habenula in aversion-resistant drinking and determine which inputs to lateral habenula from regions of the glutamatergic basal forebrain most effectively modulate compulsive alcohol consumption. Together, these experiments will yield novel insights into the neural circuits mediating compulsive alcohol use, as well as aversion-related constraint of reward-seeking more broadly.