Yanhua H. Huang - US grants

Abstract Sleep profoundly affects emotional and motivational states. Sleep disturbance is a co-morbidity in almost all emotional and motivational disorders including drug addiction, depression, and schizophrenia. Indeed, sleep disturbance is not only a symptomatic consequence, but also a causal factor for the progression of these pathological emotional states. Clinical statistics show that people with insomnia are more prone to drug addiction. Despite such apparent significance, little is known about how sleep disturbance regulates the function of key brain regions that control emotion and motivation. This application focuses on this important yet under-explored research area. The proposed research attempts to characterize how sleep deprivation (SD) regulates both excitatory and inhibitory synapses as well as the membrane properties of neurons within the nucleus accumbens (NAc), a brain region that gates emotional and motivational output. Malfunction of the NAc contributes to several pathological emotional and motivational states, such as drug addiction, anxiety, and depression. Although clinical statistics shows a clear correlation between the intensity of sleep disturbance and the degree of these emotional disorders, it remains to be determined how NAc neurons are altered in sleep- disturbed subjects. By characterizing the effect of SD on synaptic transmission and membrane properties of NAc neurons, the proposed work will provide a relatively comprehensive picture about how sleep disturbance affects the functional output of the NAc. The central hypothesis guiding the proposed research is that SD differentially regulates excitatory and inhibitory synaptic activities as well as the membrane excitability of NAc neurons, resulting in distorted functional output of NAc neurons. This hypothesis will be tested by pursuing three specific aims: 1) characterize SD-induced synaptic adaptations in NAc neurons; 2) characterize SD- induced membrane adaptations in NAc neurons; and 3) explore the impact of SD on NAc-based emotional and motivational behaviors. The proposed work is closely relevant to NIH's mission in that the expected outcome will provide essential understanding about how sleep and sleep disturbance regulate a key brain region that is involved in a large number of emotional and motivational disorders.

DESCRIPTION (provided by applicant): Endocannabinoids (eCBs) mediate one of the most prevalent and complex signaling systems in the central nervous system. They are ubiquitous regulators of excitatory and inhibitory neural transmission, and studies over the past two decades have well established a retrograde mechanism of eCB signaling. Specifically, postsynaptic neurons release eCBs, which activate presynaptically located eCB receptor type 1 (CB1R) and regulate release of neurotransmitters. This retrograde mechanism has become the central tenet of theories describing how eCBs regulate synaptic transmission, which they do at both excitatory and inhibitory synapses in many brain regions. However, we have recently acquired data from dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) that depict a scenario in distinct contrast to this dominant concept. Using optogenetic, electron microscopic and electrophysiological tools, our preliminary studies demonstrate a form of short-term synaptic plasticity that is likely triggered by eCBs released from dopaminergic axon terminals. Briefly, i) Selective activation of VTA-to-NAc dopaminergic axons triggers short- term inhibition of GABA transmission at adjacent synapses; ii) Activation of GABAergic synapses alone does not trigger this effect; iii) Inhibiting CB1Rs prevents this heterosynaptic plasticity; iv) The plasticity persists following postsynaptic chelation of Ca2+ by BAPTA, and in the presence of inotropic and metabotropic glutamate receptor blockers; v) Anandamide (AEA) is one of the two major eCBs in the CNS, and N- acylphosphatidylethanolamine phospholipase D (NAPE-PLD), an enzyme that synthesizes AEA, is identified in presynaptic terminals within the NAc. These results raise a striking possibility tat eCBs may be synthesized and released from dopaminergic presynaptic terminals and regulate adjacent GABAergic synapses in a cross- synaptic manner. This novel form of eCB-mediated cellular behavior, if verified, represents the first demonstration of presynaptic release of eCBs i the NAc. This CEBRA R21 application will more thoroughly characterize this exciting new form of eCB release. We will achieve this goal by thoroughly characterizing eCB release from VTA-to-NAc dopaminergic projections with definitive approaches, including optogenetic, pharmacological, molecular, and electron microscopic assays. Specifically, we will determine the type(s) of eCB that mediates the heterosynaptic regulation, the synthetic enzyme(s) that produces the eCB, and the pre- or postsynaptic sites where the eCB is released. The expected results will clarify whether presynaptic dopaminergic terminals do indeed release eCBs within the NAc, and thus potentially provide a new direction for understanding the cellular and behavioral roles of the eCB system. Given that eCBs are implicated in the pathophysiology of several brain diseases, including drug addiction, depression, and obesity, this line of research is highly relevant to the mission of the NIH.

DESCRIPTION (provided by applicant): Cocaine addiction remains a critical medical and social problem. A prominent guiding hypothesis for the molecular and cellular research of drug addiction is the neuroadaptation theory, which suggests that addictive drugs usurp common neural plasticity mechanisms that normally help form episodic memories to instead form addiction-related memories. Although this theory has been supported by the striking similarities between drug-induced cellular adaptations and experience-dependent neural plasticity, it falls short in explaining how addiction-related memories are extremely durable and resistant to extinction. Using cocaine as the drug model, we have begun to address this critical point over the past few years by hypothesizing that exposure to cocaine wakes up dormant, highly-efficient cellular mechanisms that are otherwise only present in the developing brain to profoundly reform specific neural circuits, resulting in extremely durable circuitry and behavioral alterations associated with addiction. This hypothesis was based on our initial observation (published in 2009) that exposure to cocaine generates a large number of silent excitatory synapses in the nucleus accumbens shell (NAc), an essential brain region for motivated behaviors. Silent synapses usually only contain NMDA receptors (NMDARs), with AMPA receptors (AMPARs) either absent or highly labile. Thus, these synapses are often silent at near resting membrane potentials. Abundant in the developing brain, many silent synapses are thought to be immature synaptic contacts; upon maturation by recruiting/stabilizing AMPARs, silent synapses may evolve into fully functional synapses to form new circuits. As such, the generation and potential maturation of silent synapses may be one of the critical developmental mechanisms that exposure to cocaine resumes to induce long-lasting circuitry and behavioral alterations. Among extensive excitatory synaptic inputs to the NAc, the afferents from the medial prefrontal cortex (mPFC) are particularly important for several core aspects of cocaine addiction including cocaine seeking and craving after withdrawal from cocaine self-administration (SA). Our current preliminary results show that silent synapses are generated within the mPFC-to-NAc pathway, and maturation of cocaine-generated silent synapses within this pathway is temporally correlated with the progressive intensification of cocaine seeking (incubation of cocaine craving). Using cocaine SA and seeking as the animal models, we will test the hypothesis that cocaine SA recruits developmental mechanisms to generate silent synapses within the mPFC-to-NAc pathway in the adult rat brain; maturation of these synapses during cocaine withdrawal and the resulting re-organization of the mPFC-to-NAc pathway critically contribute to withdrawal-associated cocaine craving and seeking. Expected outcomes of the proposed research will unveil novel molecular and cellular processes contributing to cocaine relapse and provide molecular targets for potential clinical treatment.

DESCRIPTION (provided by applicant): Timing-dependent dopamine (DA) release in the nucleus accumbens (NAc) has been critically implicated in emotion-cognition interaction as well as reward-associated learning. A shift in the timing of DA release in the NAc has been shown to accompany different stages of reward conditioning. However, despite its apparent physiological significance, the cellular mechanisms by which timing-contingent DA release regulates NAc synaptic transmission have not been directly assessed. To address this knowledge gap, this application focuses on the excitatory synaptic input from the ventral medial prefrontal cortex (mPFC) to the nucleus accumbens shell (NAcSh), a key brain site involved in the integration and expression of emotional and motivational behaviors. Our preliminary results show that mPFC-to-NAcSh synaptic transmission undergoes long-term potentiation (LTP) by synchronized dopamine signaling from the ventral tegmental area (VTA). Our central hypothesis is that this LTP is DA timing-dependent and glutamate pathway-specific; it is achieved by augmented presynaptic glutamate release, and is regulated by co-activation of presynaptic dopamine and GABA receptors. To test this hypothesis, we will use whole-cell patch-clamp techniques combined with pharmacological, optogenetic, and viral-mediated gene transfer to determine the molecular and cellular mechanisms that underlie this novel form of LTP. Results from the proposed experiments will advance our knowledge of timing-dependent DA action in regulating synaptic plasticity, provide a mechanistic understanding of the mPFC-VTA-NAcSh circuitry mechanisms underlying emotion-cognition interactions, and identify molecular targets for the clinical corrections of certain cognitive, psychological, and psychiatric disorders.

Abstract Functional dissection of neural networks has been revolutionized by the introduction of optogenetic and chemogenetic tools. However, the specificity that can be achieved by currently available tools often falls short, particularly for synapse-specific manipulations. Specifically, although conventional optogenetic and chemogenetic tools are readily applicable to manipulate synaptic transmission by targeting selective subsets of presynaptic inputs or postsynaptic targets, it is often challenging to target synapses based on the unique combinatorial identity of presynaptic inputs and postsynaptic targets. One prominent example is in the nucleus accumbens (NAc), where excitatory glutamatergic inputs from multiple brain regions converge onto principal medium spiny neurons (MSNs). MSNs that express dopamine D1 or D2 receptors (D1 vs. D2 MSNs) receive shared inputs, but drive distinct cellular networks and differentially regulate reward processing. A much desired research tool is to manipulate synaptic transmission at selective subsets of presynaptic inputs onto highly defined postsynaptic neurons (e.g. D1 vs. D2 MSNs). To meet this great challenge, we tested a prototype ?trans-synaptic luminescent opsin? (tsLMO) as a novel approach for highly specific and versatile manipulations of synaptic transmission. TsLMOs consist of postsynaptic luciferase and presynaptic opsins matched to the bioluminescence wavelengths of the luciferase. Upon substrate application, bioluminescence in the post- synaptic neuron acts trans-synaptically to activate the presynaptic opsins, which in turn regulate transmitter release. Thus, the independent expression of the two molecular components of tsLMOs allows for unprece- dented high specificity and versatility for manipulations of synaptic transmission. We have successfully verified the initial efficacy of tsLMOs-based manipulation and will apply it to NAc circuits. We will target the rostral basal lateral amygdala (rBLA)-to-ventral lateral NAc (vlNAc) projection, a subset of the BLA-to-NAc inputs, whose function has not been well understood. Our preliminary results show that this projection regulates natural reward seeking in mice in manners that contrast with current literature. Furthermore, the rBLA-to-vlNAc projection makes monosynaptic excitatory inputs onto both D1 and D2 MSNs, and the two subsets of synapses show differential regulation of glutamate release under physiological conditions. Thus, we propose to use tsLMOs to determine whether rBLA-to-vlNAc transmission onto vlNAc D1 and D2 MSNs differentially regulates natural reward seeking. Expected outcome of this application includes (i) successful development of synaptically targeted tsLMOs for highly specific and versatile manipulations of synaptic transmission; and (ii) an example application of the tsLMO approach for dissecting a specific NAc circuit in reward-elicited behaviors. Given the unique advantages of the tsLMO approach in specificity, versatility, and feasibility, it will become an indispensible tool for functional dissection of neural networks in current neuroscience research. Thus, this application fits the goal of the CEBRA R21 mechanism, and is highly relevant to the mission of the NIH.

Abstract Sleep abnormalities commonly occur among chronic cocaine users long after withdrawal. The withdrawal- associated sleep problems, including loss of sleep and worsened sleep quality, have been speculated to foster cocaine use and relapse. However, it is not well understood whether and how sleep-associated mechanisms may regulate the brain reward circuitry and impact relapse-like behaviors. In rats withdrawn from cocaine self- administration, there is persistent reduction in total sleep time and increased sleep fragmentation, resembling the changes in humans. Interestingly, experimentally increasing REM (without changing NREM) sleep after withdrawal reduces cocaine craving, suggesting REM sleep-associated mechanisms in this regulation. In the nucleus accumbens (NAc), a key brain region for reward processing, there is progressive accumulation of the GluA1-rich, calcium-permeable, AMPA receptors (CP-AMPARs) after long-term withdrawal from cocaine. These receptors critically contribute to the intensified cue-induced cocaine craving after long-term withdrawal. Importantly, sleep intervention that increases REM sleep time/duration leads to decreased CP-AMPARs in the NAc, which is accompanied by reduced cocaine craving after withdrawal. These results not only suggest NAc CP-AMPARs as key neuronal substrates that express sleep-mediated regulation of cocaine craving and relapse, but raise the important question ? how does REM sleep-associated mechanisms route to NAc CP- AMPARs? Accumulating evidence suggests that the melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus (LH) may critically contribute to the REM sleep-mediated effects. Activity of LH MCH neurons during sleep drives the initiation and/or maintenance of REM sleep. Moreover, MCH neurons project to the NAc, where MCH receptors are highly expressed; MCH receptor signaling in the NAc strongly regulates the phosphorylation and synaptic removal of GluA1-containing AMPARs. Preliminary results further show that LH MCH neurons exhibited reduced membrane excitability long after withdrawal from cocaine, whereas mimicking MCH release in the NAc after long-term withdrawal by intra-NAc infusion of MCH during the light (sleep) phase led to reductions of CP-AMPARs in the NAc and reduced cocaine craving during the subsequent dark (active) phase. Together, these results suggest that an increase in the activity of MCH neurons may be critical for both normalizing REM sleep and reducing cocaine craving after withdrawal. This application aims to test the hypothesis that enhancing the activity of LH MCH neurons during sleep will increase REM sleep time/duration and reduce cocaine craving after withdrawal. Expected results of the proposed research will identify a putative REM sleep-associated mechanism that modulates synaptic transmission within the brain reward circuitry and regulates cocaine craving after withdrawal. Given the close association between sleep disturbance and the co-morbid psychiatric diseases including drug addiction and depression, this application is highly relevant to the mission of the NIH.

Abstract Sleep dysregulation is a hallmark of alcohol use disorders (AUDs) and disrupted sleep can contribute to relapse even after months of abstinence. Despite the well-recognized sleep-AUD interactions, few studies have investigated how sleep changes over the development of excessive drinking, and the mechanisms by which sleep disruptions exacerbate drinking and/or relapse liability are largely unknown. Based on several key findings from published studies and preliminary work, one hypothesis is that disrupted REM sleep induces maladaptive changes in the medial (M) and lateral habenula (LHb), which in turn contribute to escalated alcohol intake and promote relapse. The key findings are: 1. Specific REM sleep deficits at 2-3 weeks after alcohol withdrawal are a robust predicting factor for relapse in alcohol dependent patients; 2. Chronic, selective REM sleep fragmentation enhances medial Hb neuron tonic firing and negative affect; 3. Relapse to alcohol seeking is associated with increased activation of MHb and LHb neurons. 4. Sleep fragmentation precipitates drug craving during abstinence from cocaine, while selective consolidation of REM sleep reduces craving. Thus, it is predicted that chronic alcohol drinking and abstinence leads to disrupted REM sleep, which induces Hb hyperactivity, promoting a negative affective state that drives motivation for alcohol and relapse. Likewise, it is predicted that chronic REM sleep disruptions can initiate this cycle, leading to increased risk for developing AUDs. Therefore, aim 1 in this proposal will evaluate the effects of chronic and escalating alcohol drinking and abstinence on sleep architecture and Hb neural activity. The proposed experiments will use the intermittent access two-bottle choice ethanol drinking paradigm combined with chronic EEG/EMG recordings and ex vivo slice electrophysiology. It is expected that the baseline REM sleep features or the REM sleep deficits following alcohol drinking/abstinence may predict the amount of alcohol drinking as well as Hb neural activity. Aim 2 will determine if chronic REM sleep fragmentation promotes the escalation of alcohol intake in the intermittent access model and/or promotes relapse in an operant self-administration model. We will further determine if the REM sleep effects on alcohol intake/relapse are, in part, mediated by REM sleep fragmentation-induced hyperactivity of Hb neurons. Aim 3 will test the treatment potential of interventions that improve REM sleep. Utilizing recently developed REM sleep-selective manipulations, it will be determined if selective REM sleep consolidation can reduce alcohol drinking and relapse, and if this is associated with reduced Hb activity. Overall, the proposed studies will provide a critical assessment of the potential for developing REM sleep- focused therapeutics for preventing relapse, and provide novel mechanistic insight into the interactions between alcohol intake and sleep homeostasis in the context of habenula dysregulation. Thus, this proposal is consistent with the mission of NIAAA RFA-AA-19-006 to perform Mechanistic Studies on Chronic Alcohol Use and Sleep Homeostasis.

Abstract Sleep abnormalities commonly occur among chronic cocaine users after withdrawal, including loss of total sleep time and increase in sleep fragmentation. The withdrawal-associated sleep problems are speculated to foster cocaine use and relapse, however, whether and how sleep mechanisms may regulate the brain reward circuitry and impact relapse-like behaviors remain elusive. Using a rat cocaine self-administration model to recapitulate sleep loss and fragmentation after withdrawal, the Huang lab has obtained direct evidence of sleep-induced regulation of cocaine seeking: experimentally increasing REM (without changing NREM) sleep episode durations reduces cocaine seeking after withdrawal, suggesting REM sleep-associated mechanisms in this regulation. Neural mechanism studies have focused on the nucleus accumbens (NAc), a key brain region for reward processing. Progressive accumulation of the GluA1-rich, calcium-permeable, AMPA receptors (CP- AMPARs) at synapses in the NAc critically contributes to the intensified cocaine seeking after withdrawal. Importantly, behavioral sleep interventions that increase REM sleep episode durations lead to decreased accumulation of NAc synaptic CP-AMPARs. These results not only suggest NAc CP-AMPARs as key neuronal substrates that express REM sleep-induced anti-relapse effects, but raise the critical question ? how do REM sleep interventions route to NAc CP-AMPARs? Published and preliminary results suggest that the melanin- concentrating hormone (MCH) neurons in the lateral hypothalamus and zona incerta (LH for short) may play an important role in the REM sleep-induced anti-relapse effects. LH MCH neurons predominantly fire during REM sleep. Behaviorally induced sleep rebound after REM sleep restriction, a strategy utilized by our sleep intervention, further enhances the activity of these neurons. Moreover, MCH neurons project to the NAc, where MCH receptors (MCHRs) are highly expressed; MCHR signaling in the NAc strongly regulates the phosphorylation and facilitates synaptic removal of GluA1-containing AMPARs. Preliminary results further show that LH MCH neurons exhibited reduced membrane excitability after withdrawal from cocaine, whereas mimicking MCH release by intra-NAc infusion of MCH during light (sleep) phase led to reduction of synaptic CP-AMPARs in the NAc and decreased withdrawal-associated cocaine seeking. Together, these results suggest that REM sleep interventions may engage LH MCH neural activity to produce anti-relapse effects after withdrawal. This application will test the hypothesis that MCH signaling during sleep contributes to REM sleep-induced anti-relapse effects after withdrawal from cocaine. In contrast to the current practice focusing on NREM sleep, this proposal will identify a novel REM sleep mechanism that produces anti-relapse effects. Moreover, the proposal emphasizes target manipulations during sleep rather than in wakefulness. Concerning the high comorbidity between drug withdrawal and sleep disturbance, this application reveals novel strategies for developing sleep-based therapies for drug addiction, thus is highly relevant to NIH?s mission.

PROJECT SUMMARY  Adolescence is a vulnerable period for initiating substance use and abuse, during which time sleep and circadian  rhythm  disruptions  are  pervasive.  It  is  increasingly  recognized  that  sleep  and  circadian  rhythm  causally,  and  powerfully regulate reward processing, but the underlying mechanisms remain poorly understood. Could sleep  and  circadian  rhythm  traits  be  related  to  reward  circuit  function?  Whether  and  how  do  sleep  and  circadian  disruptions lead to increased vulnerability for substance use in adolescents? The central hypothesis of the Center  application  is  that adolescent development acts  on underlying  sleep and circadian  traits  to  modify homeostatic  sleep  drive,  circadian  phase,  and  circadian  alignment,  which  in  turn  impact  cortico-­limbic  functions  critical  to  substance  use  risk  (e.g.,  reward and  cognitive  control). It  is  further hypothesized that  specific  manipulations of  sleep  and  circadian  rhythms  during  adolescence  will  affect  reward  responsivity  and  cognitive  control  in  either  positive  or  negative  directions.  This  research  project  (Project  5)  will  focus  on  rodent  models  to  determine  the  cellular and synaptic mechanisms within the cortico-­limbic circuit through which sleep and circadian disruptions  alter reward processing. Specifically, the nucleus accumbens (NAc) is a reward-­processing ?hub? in the ventral  striatum which is sensitive to both sleep and circadian disruptions. For example, acute sleep deprivation reduces  glutamate release at medial prefrontal cortex-­to-­NAc medium spiny principal neurons (MSNs) synapses;? chronic  sleep  fragmentation  increases  cholinergic  neural  activity  in  the  NAc  (preliminary  results);?  robust  diurnal  fluctuations in AMPA receptor (AMPAR) levels and intrinsic membrane excitability are also observed in the NAc  MSNs, and circadian gene mutation in the NAc leads to altered AMPAR transmission in MSNs. Together, these  results suggest that the NAc may represent a converging site for sleep and circadian rhythm to regulate reward  processing. Accordingly, Project 5 will test the hypothesis that sleep and circadian rhythm target aspects of NAc  synaptic  transmission  and  neural  modulation  to  regulate  reward-­motivated  behaviors.  Thus,  Aim  1  will  use  genetically  diverse  outbred  rats  to  determine  whether  naturally  occurring  ?early?  and  ?late?  chronotypes  are  associated  with  different  diurnal  variation of  AMPAR  transmission  in  NAc  MSNs.  This  aim  will  accommodate  molecular  (Project 3) and  behavioral  (Project  4) characterizations  of  these  rats.  Aim 2  will  determine  whether  circadian  disruptions  without  changes  in  sleep  alter  the  diurnal  variation  of  membrane  excitability  and/or  postsynaptic  AMPAR  levels  in  the  NAc  MSNs.  Aim  3  will  determine  the  effects  of  acute  and  chronic  sleep  restrictions on adenosine and cholinergic transmission in the NAc, and the behavioral consequences in natural  or drug self-­administration. The expected outcome of Project 5 will integrate and extend findings from behavioral  (Project 4) and molecular genetic (Project 3) studies, which together will provide mechanistic insights to inform  and further develop human studies (Projects 1&2).