Yan Dong - US grants

[unreadable] DESCRIPTION (provided by applicant): Cancer prevention is the most effective strategy to reduce cancer morbidity and mortality. Therefore, it is imperative to develop new investigators in this area of research. Yan Dong, a junior investigator in the field of cancer chemoprevention, has extensive training in molecular biology and cell biology. The support from this award will foster Dr. Dong's development as an independent researcher in the field of human cancer prevention. This project will focus on the investigation of molecular mechanism of selenium chemoprevention of prostate cancer. A previous randomized, placebo-controlled cancer prevention trial showed that selenium supplementation significantly reduced the incidence of prostate cancer, although the mechanism of action is still unknown. The proposed research will test the hypothesis that selenium prevents prostate cancer by suppressing androgen receptor (AR) signaling. The research plan consists of four aims. Aim 1 is to study the mechanism of reduced AR transcription by selenium using the reporter gene assay, mutagenesis analysis, and EMSA. Aim 2 is to investigate selenium-modulation of AR ligand binding, dimerization, and nuclear translocation by the whole-cell radioligand binding assay, protein-protein interaction analysis with a mammalian two-hybrid system, as well as Western and immunofluorescence analyses, respectively. Aim 3 is to assess the functional significance of AR downregulation in selenium chemoprevention of prostate cancer. This issue will be addressed both in vitro and in vivo using stable AR transfectants. Aim 4 is to characterize AR-mediated gene expression changes critical for selenium anticancer effect by the real-time RT-PCR analysis in both in vitro and in vivo models. The overall goal of this project is to sharpen Dr. Dong's scientific and technical acumen essential for carrying out translation research and obtain R01 support for the establishment of an independent research program directly relevant to the prevention of human cancer. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The leading hypothesis in addiction research is that exposure to drugs of abuse induces adaptive neu- ronal changes, resulting in addictive behaviors. The many experiments conducted on the basis of this neuro-adaptation theory have identified a huge number of drug-induced cellular changes related to addic- tion. For clinical treatment, however, it is impossible to directly manipulate each of these changes. Our long-term research goal is, thus, to identify the molecular `controllers'that trigger and maintain drug-induced neural adaptations;manipulations of these key molecules may then collectively correct other subordinate pathophysiological cellular changes. This proposal focuses on the N-methyl-D-aspartate receptor (NMDAR), a key molecule that governs multiple forms of neural plasticity and that is a potential molecular controller of addiction-related neural adaptations. Our preliminary studies show that cocaine exposure persistently alters the function of NMDARs in nucleus accumbens (NAc) neurons;experimentally mimicking this change of NMDARs triggers secondary cellular adaptations related to addiction. We hypothesize that this cocaine- induced NMDAR adaptation steers a collection of NMDAR-dependent cellular processes toward addiction- specific adaptations. In this application, we propose an extensive but realistic set of experiments to (1) further characterize cocaine-induced adaptation in NAc NMDARs, (2) examine the underlying molecular mechanisms, and (3) investigate the cellular consequences. To achieve these goals we will use a multi- disciplinary approach utilizing patch-clamp recordings, viral-mediated gene transfer, biochemical assays, and behavioral tests. Relevance to Public Health: By characterizing this novel NMDAR adaptation, our proposed study will define a potential molecular trigger for persistent cocaine-induced adaptations, thus providing relevant mechanistic insights to underpin advances in prevention and treatment of addiction. PUBLIC HEALTH RELEVANCE Project Narrative: The proposed studies will characterize a key molecule that potentially controls a large collection of cocaine-induced, addiction-related neural adaptations. Results from our proposed research will have significant impact on public health because once this `controlling molecule'is defined, therapeutic strategies can be designed accordingly to correct a great number of cocaine-induced cellular adaptations. As such, the findings are expected to lead to novel and effective treatments for human addiction.

DESCRIPTION (provided by applicant): Drug addiction can be conceptualized as an extreme form of memory, a memory that is distinctly long-lasting and completely new for individuals who have never experienced drugs of abuse. Whereas modification of pre- existing synapses/neural circuits has been widely accepted as a cellular mechanism for forming memories, we hypothesize that drug addiction as a new and extremely robust form of memory is mediated not only by modification of the existing synapses, but also by the formation of new synaptic connections and thus new neural circuits. This hypothesis, although seemingly striking, is consistent with several lines of evidence showing that following exposure to cocaine or other addictive psychostimulants, new dendritic spines and premature excitatory synaptic connections are formed in the nucleus accumbens (NAc), a critical brain region for the development of drug addiction. To directly measure the potential new synaptic connections/neural circuits induced by exposure to cocaine, we propose to adapt and develop an innovative imaging technique, the GRASP (GFP reconstitution across synaptic partners) technique, which can label newly formed synaptic connections in vivo. Our strategy is to split GFP into two halves, one expressed in potential presynaptic terminals and the other in potential postsynaptic terminals. These two halves do not fluorescence individually but will reconstitute into a fluorescent GFP when the pre- and post-synaptic terminals interact to form new synapses. The proof-of-concept data from our preliminary studies suggest that this experimental strategy is highly promising. In this application, our first objective is to optimize the GRASP technique such that it can be readily used for in vivo labeling of new excitatory synapses. Our second objective is to use this technique to characterize a potential new neural projection to the NAc following cocaine exposure. Using a Fluoragold- based tracing approach, our preliminary data showed that following exposure to cocaine, the NAc received new, intense innervations from the lateral habenula, a projection that was not observed in saline-treated control animals. Thus, the proposed work will not only develop an in vivo GRASP technique, but also use this technique to address an important neuroscience question. As such, this proposal is consistent with the mission of CEBRA application, and the outcome of this proposal will provide the field with a novel in vivo technique and a potentially novel circuitry-based mechanism underlying cocaine addiction. PUBLIC HEALTH RELEVANCE: This application will optimize a novel investigating technique that can be used to characterize newly formed synaptic connections upon in vivo experience. With this technique, we will also determine a potential newly formed neural circuit within the nucleus accumbens upon exposure to cocaine. The proposed work will not only develop/optimize an innovative in vivo technique, but also open a new avenue toward understanding circuitry- based mechanisms for drug addiction.

DESCRIPTION (provided by applicant): The HIV-associated emotional and motivational disorders (HEMDs) include severe depression, serious apathy, persistent sadness, and decreased appetite. These HEMDs cause non-compliance with treatment, an increase in high-risk behaviors, and suicidal death, and thus substantially impact the already-afflicted life of AIDS-patients. A gap in our knowledge that prevents us from developing effective treatments for HEMDs is that we do not know the neuronal mechanisms that mediate HEMDs. To address this knowledge gap, we will target the nucleus accumbens (NAc) because this site is one of the central targets for HIV-infection, and malfunction of the NAc results in a variety of emotional and motivational disorders similar to HEMDs. Our longterm goal is to identify the key molecular substrates in the NAc, the manipulation of which can prevent or ameliorate HEMDs. Our promising preliminary results suggest that the NR2A-containing N-methyl-Daspartate receptor (NMDAR) and its coupled signaling may be such key molecular substrates that can protect NAc neurons from the HIV insult. As an initial step toward our long-term goal, the objective of this R21 application is to 1) establish the relationship between HIV-induced NAc neurodegeneration and motivational deficits; and 2) determine the potential neuroprotective effect of manipulating the NMDAR-pathway in HIVinduced NAc neurodegeneration in a Tat-expressing mouse line. Tat is a neurotoxic protein released by HIV. Our central hypothesis is that Tat-induced NAc neurodegeneration is positively correlated with compromised motivational behaviors, and that activation of NR2A-containing NMDARs protects Tat-induced neurodegeneration of NAc neurons. Given that several NMDAR-based compounds have already been used in clinical trials, our proposed research may have immediate clinical impact on the treatment of HEMDs. Thus, our studies are highly relevant to the mission of the NIH to develop fundamental knowledge that will potentially help to reduce the burden of human disability. Guided by our promising preliminary data, our objective will be achieved by pursuing two specific aims: 1) Define the role of Tat-induced NAc neurodegeneration in HEMDs; and 2) Define the neuroprotective role of the NR2A-pathway in Tat-induced NAc neurodegeneration. Under both aims, we will use a line of transgenic mice in which expression of Tat can be temporally and quantitatively controlled. The proposed work is innovative because it will establish the first HEMD model linking the HIV-induced cellular and molecular changes to a detectable behavioral output. The proposed work also broadly and positively impacts the NeuroAIDS field as a whole in that the advanced behavioral and electrophysiological paradigms to be established are broadly applicable to studies of other HIV-induced neurodegenerative mechanisms. Furthermore, the NMDAR-based mechanism to be examined may have a general neuroprotective effect in other HIV-induced pathological conditions.

Abstract During drug abstinence, re-exposure to cues previously associated with cocaine often trigger drug relapse. In rodent models of cocaine seeking and relapse, animals that self-administer cocaine in the presence of contingent cues often establish a strong association between cues and cocaine, such that after drug withdrawal, the presence of cues induces strong cocaine seeking. Cue-induced cocaine seeking is extremely long-lasting and intensifies progressively after withdrawal. The goal of this application is to develop new concepts and approaches through which the cue-cocaine association can be disrupted to reduce cocaine relapse. The cue-cocaine association that drives cocaine seeking shares general features of cue-conditioned memories. Similar to classic conditioning memories, cue-conditioned drug memories also undergo a destabilization and reconsolidation process after retrieval. During the brief destabilization time window, amnesic treatments are often more effective in reducing subsequent cue-induced cocaine seeking. However, the neural substrates that mediate cue-drug memory retrieval and reconsolidation remain elusive. Targeting this knowledge gap, this application focuses on cocaine-generated silent synapses and their dynamic changes within the basolateral amygdala (BLA) to nucleus accumbens (NAc) projection. We recently showed that cocaine self-administration generates silent synapses in the BLA-to-NAc projection. Silent synapses are excitatory synapses that contain NMDA receptors without stable AMPA receptors (AMPARs). Our additional results suggest that cocaine-generated silent synapses may serve as the initial hubs to establish a potentially new set of circuits. After cocaine withdrawal, BLA-to-NAc silent synapses become `un-silenced' by recruiting calcium-permeable AMPARs (CP-AMPARs), resulting in consolidation of the silent synapse-imbedded circuits. Reversing the un-silencing of BLA-to-NAc silent synapses decreases cue-induced cocaine seeking. These results suggest that the newly formed, silent synapse-embedded BLA-to-NAc projections contribute to the establishment and subsequent consolidation of cue-cocaine association. Based on extensive preliminary results, we hypothesize that after cocaine withdrawal, a brief re-exposure to cocaine-associated cues instantly induces CP-AMPAR internalization and re-silences the same set of BLA-to-NAc silent synapses that are generated by cocaine self-administration, contributing to the destabilization of cue-cocaine association. These re-silenced synapses are un-silenced again by re-recruiting CP-AMPARs hours after cue re-exposure, contributing to reconsolidation of cue-cocaine association. We will use electrophysiology, optogenetics, in vivo calcium imaging, viral-mediated gene transfer, and operant behavioral assays to test this hypothesis. By accomplishing the proposed work, we may identify a cellular and circuit basis underlying destabilization and reconsolidation of cue-cocaine association after retrieval and validate therapeutic angles to reduce cocaine relapse. Thus, objectives of this application are highly relevant to the missions of the NIDA, NIH.

DESCRIPTION (provided by applicant): Cues associated with prior cocaine use are powerful triggers of relapse in abstinent cocaine users and of drug seeking in cocaine-experienced rodents. Rodent studies show that cue-induced cocaine craving progressively intensifies over the course of withdrawal from extended access cocaine self-administration. This phenomenon, known as incubation of cocaine craving, may contribute to the difficulty of maintaining abstinence from cocaine use. Growing evidence supports the relevance of incubation to drug craving in humans. A key feature of the incubation process is that, once initiated, it continues to exacerbate automatically during the withdrawal period, without apparent external stimulation. This suggests the involvement of homeostatic rather than Hebbian forms of neuronal plasticity. Using a mouse model, this proposal aims to determine the role of homeostatic plasticity in the nucleus accumbens (NAc), a key brain region for addiction, in the incubation of cocaine craving. Ho- meostatic plasticity is a physiological self-correcting mechanism through which neurons compensate for 'unde- sirable' cellular alterations, thus stabilizing their functional output. Are there any forms of homeostatic neural plasticity in NAc neurons that may help these neurons regain normal function following cocaine exposure? We previously demonstrated a form of homeostatic crosstalk between excitatory synaptic input and intrinsic mem- brane excitability in NAc neurons. This phenomenon, termed homeostatic synapse-membrane crosstalk (HSMC), enables NAc neurons to adjust their intrinsic membrane excitability to functionally offset alterations in excitatory synaptic strength. As a consequence, the optimal output of NAc neurons may be stably maintained. However, if misled by false homeostatic signals, HSMC may be erroneously engaged, triggering cascades of homeostatic dysregulation that progressively shift neuronal output further and further from the normal set-point. In previous work, we showed that cocaine exposure increases synaptic levels of NR2B-containing NMDA re- ceptors in the NAc. Our central hypothesis, based on extensive preliminary results, is that this constitutes a false homeostatic signal that triggers HSMC and subsequent homeostatic dysregulation cascades, ultimately resulting in a persistent decrease in membrane excitability and an increase in synaptic strength. Together, these changes are hypothesized to magnify the response of NAc neurons to cocaine-associated cues and the- reby elicit incubation of cocaine craving. To test this hypothesis, this proposal will characterize key molecular substrates for HSMC-based dysregulation cascades (e.g., glutamate receptors and SK-type potassium chan- nels), examine the role of dopamine in modulating these cascades, and develop a HSMC-based approach to attenuate incubation of cocaine craving. To achieve these goals, we will use a multidisciplinary approach com- bining in vivo molecular/pharmacological manipulations, biochemistry, slice electrophysiology, and behavioral tests. Our results will set the stage for translational studies aimed at developing a homeostasis-based pharma- cological strategy to restore normal NAc function in cocaine users.

DESCRIPTION (provided by applicant): Exposure to drugs of abuse causes dysfunction of nucleus accumbens (NAc) neurons, which are strongly linked to motivation and addiction. Despite the estimate that an enormous number of drug-induced effects represent homeostatic responses, drug-induced homeostatic regulation and dysregulation in the NAc have not been well characterized. This application will explore the contribution of homeostatic plasticity to cue induced cocaine craving. More specifically, it has been observed that cues associated with prior cocaine use are powerful triggers of relapse in abstinent cocaine users and of drug seeking in cocaine-experienced rodents. This cue-induced cocaine craving progressively intensifies (incubates) over the course of withdrawal from extended access cocaine self-administration. Growing evidence supports the relevance of incubation to drug craving in humans. A key feature of the incubation process is that, once initiated, it continues to exacerbate automatically during the withdrawal period, without apparent external stimulation. This suggests the involvement of homeostatic rather than Hebbian forms of neuronal plasticity. Using rats, we propose to determine the role of homeostatic plasticity in the NAc in the incubation of cocaine craving. Homeostatic plasticity is a physiological self-correcting mechanism through which neurons compensate for 'undesirable' cellular alterations, thus stabilizing their functional output Are there any forms of homeostatic regulation/dysregulation in NAc neurons that may be involved in incubation of craving? We previously demonstrated a form of homeostatic crosstalk between excitatory synaptic input and intrinsic membrane excitability in NAc neurons. This phenomenon, termed homeostatic synapse-membrane crosstalk (HSMC), enables NAc neurons to adjust their intrinsic membrane excitability to functionally offset alterations in excitatory synaptic strength. As a consequence, the optimal output of NAc neurons may be stably maintained. However, if misled by false homeostatic signals, HSMC may be erroneously engaged, triggering cascades of homeostatic dysregulation that progressively shift neuronal output further and further from the normal set-point. Our central hypothesis, based on extensive preliminary results, is that increased transmission via NR2B-containing NMDARs constitutes a false homeostatic signal that triggers HSMC and subsequent homeostatic dysregulation cascades, ultimately resulting in a persistent decrease in membrane excitability and an increase in synaptic strength. Together, these changes are hypothesized to magnify the response of NAc neurons to cocaine-associated cues and thereby contribute to incubation of cocaine craving. To test this hypothesis, this proposal will characterize key molecular substrates for HSMC-based dysregulation cascades and test the ability of HSMC-based approaches to attenuate incubation of cocaine craving. We will use a multidisciplinary approach combining in vivo molecular/pharmacological manipulations, biochemistry, slice electrophysiology, and behavioral tests. Our results will set the stage for translational studies aimed at developing a homeostasis-based pharmacological strategy to restore normal NAc function in cocaine users.

DESCRIPTION (provided by applicant): The long-term objectives of this application are to understand the molecular mechanisms of resistance to androgen-directed therapies and to develop effective strategies to overcome the resistance. Recent significant advances in our understanding of continued androgen receptor (AR) signaling in castration-resistant prostate cancer have led to the development and FDA approval of two next-generation androgen-directed therapies, abiraterone and enzalutamide (MDV3100), which heralded a new era of prostate cancer therapy. However, disease progression after androgen-directed therapies remains the most critical challenge in the clinical management of prostate cancer. Upregulated expression of constitutively-active, alternatively-spliced AR variants (AR-Vs) that lack the ligand binding-domain has been implicated to play an important role in disease progression. However, the precise mechanism by which androgen-directed therapies increase AR-V expression and the precise mechanism by which AR-Vs regulate target-gene expression in mediating therapeutic resistance remain poorly understood. These are two issues vital to understanding resistance to androgen-directed therapies and developing effective means to overcome the resistance, and therefore are the main focus of investigation of the proposed study. Based on the findings from ours and others, we propose that upregulation of AR-Vs is an inevitable response of prostate cancers to all androgen-directed therapies currently accepted in the clinic and that a shift from androgen-dependent full-length AR (AR-FL) homodimerization to androgen-independent AR-FL/AR-V and AR-V/AR-V dimerization leads to unleashed AR activity, driving disease progression. Given the potency of abiraterone and enzalutamide and the evidence for ongoing suppression of androgen synthesis at the time of progression on abiraterone, this resistance mechanism may be especially critical for abiraterone and enzalutamide. Three specific aims are proposed to test the hypotheses. Aim 1: Delineate the mechanism of AR-V induction following androgen-directed therapies. Aim 2: Elucidate the mechanism by which AR-Vs regulate target-gene expression. Aim 3: Correlate AR-V expression in circulating tumor cells with abiraterone/enzalutamide response. The proposed research is relevant to public health because it addresses a resistance mechanism that is inherent to all current androgen-directed therapies, including the new drugs abiraterone and enzalutamide. Moreover, we hope to establish AR-Vs as a prognostic marker to guide the selection of patients who will likely benefit from abiraterone and enzalutamide therapies. Overall, the outcome of this study is expected to have a direct impact on rational drug design and combination to overcome the shortcoming of current therapies and on reducing suffering and improving quality of life of the patients.

? DESCRIPTION (provided by applicant): In many brain regions, glial cells substantially outnumber nerve cells, but their role in physiological and pathophysiological conditions remains poorly understood. Astrocytes are the most widely distributed glia with intimate anatomical interactions with excitatory synapses. Recent studies reveal that, in addition to providing structural and nutritional support, astrocytes dictate synapse formation and subsequent synapse refinement- elimination in the developing CNS. Our preliminary results show that, after chronic exposure to cocaine or morphine, some of these glia-based developmental mechanisms re-emerge in the adult nucleus accumbens (NAc), a forebrain region essential for addiction-related behavioral abnormalities. These drug-induced, glia- mediated synaptic remodeling processes may profoundly rewire the neurocircuits involving the NAc, and critically contribute to the pathophysiology of drug addiction. Focusing on this unique angle, the objectives of this application are: 1) To characterize the molecular and cellular mechanisms underlying glia-mediated synaptogenesis and synaptodegeneration in the NAc in mice after cocaine or morphine self-administration and withdrawal; 2) To determine the circuitry consequences of drug-induced, glia-mediated synaptic remodeling, particularly, how NAc excitatory synapses are refashioned in cocaine- and morphine-exposed mice by glia- mediated synaptogenesis or synaptodegeneration; and 3) To determine the behavioral consequences of drug- induced, glia-mediated synapse and circuitry remodeling using the mouse model of incubation of cue-induced drug craving, a drug relapse model that depends on NAc excitatory circuits. To achieve these goals, we will use a multidisciplinary approach, across the Dong and Nestler laboratories, including confocal imaging, slice electrophysiology, optogenetics, in vivo viral-mediated gene transfer, RNA interference, transgenic mouse lines, and mouse models of drug self-administration. By targeting the previously unexplored glia-mediated synapse and circuitry remodeling in drug-exposed mice, the proposed experiments promise to open new avenues toward understanding cellular and circuitry mechanisms underlying drug addiction and providing new strategies for anti-addiction treatments.

Abstract The ventral tegmental area (VTA) sends dense projections to the nucleus accumbens (NAc), constituting the backbone of the mesolimbic system. The mesolimbic system is often targeted by drugs of abuse, stress, and other severe experience to change the emotional and motivational states, resulting in a variety of psychological and psychiatric disorders. In addition to dopamine and GABA, the VTA-to-NAc projections also release glutamate. The VTA-to-NAc glutamatergic transmission has been thought to critically regulate NAc principle medium spiny neurons (MSNs), and contribute to motivated behaviors as well as behavioral alterations after exposure to drugs of abuse. However, despite extensive exploration, only weak cellular effects of the VTA-to- NAc glutamatergic signaling have been detected. For example, VTA-released glutamate evokes EPSCs in NAc MSNs as well as in interneurons, but these EPSCs are usually very small compared to EPSCs evoked from other major glutamatergic sources. A critical question is whether there are cellular mechanisms through which the VTA-to-NAc glutamatergic transmission effectively regulates NAc MSNs and NAc-based behaviors. Our preliminary results reveal a novel and robust cellular role of the VTA-to-NAc glutamatergic projection. Specifically, activation of the VTA-to-NAc glutamatergic projection transiently inhibited the responsiveness of MSNs to other major excitatory inputs, providing a potential time-locked shunting of NAc MSNs upon VTA activation. Thus, rather than a depolarization driver, the VTA-to-NAc glutamatergic projection functions as a regulator of other ongoing excitatory inputs to NAc MSNs. This regulation can be particularly important in behaving animals, in which activation of NAc MSNs must be finely controlled in response to incoming excitatory inputs to achieve select behaviors. Our subsequent preliminary results suggest a novel mechanism mediating this regulation, a mechanism involving activation of NAc astrocytes and astrocytic release of gliotransmitters. Because each astrocyte ensheathes a population of neurons, this potential effect of astrocytes may help synchronize population activities of NAc MSNs. The objective of this application is to thoroughly characterize the cellular basis of this VTA-to-NAc glutamatergic transmission-mediated regulation of NAc MSNs. Based on extensive preliminary results, we hypothesize a neuron-glia-neuron interaction mechanism: activation of the VTA-to-NAc glutamatergic projection activates metabotropic glutamate receptor 5 (mGluR5) on NAc astrocytes, which results in astrocytic release of ATP to presynaptically inhibit ongoing excitatory synaptic transmissions to NAc MSNs from other major glutamatergic inputs. By testing this hypothesis, we expect to characterize the cellular and circuitry mechanisms underlying VTA-to-NAc glutamatergic regulation. These mechanisms and involved molecular substrates may provide a conceptual and experimental foundation for future comprehensive studies to reveal the cellular, circuitry, and behavioral roles of VTA-to-NAc glutamatergic signaling, and how to manipulate this signaling to achieve clinical benefits.

Abstract Drug addiction has been conceptualized as the endpoint of cascades of transitions from initial voluntary and limited drug use to habitual and escalated drug use, and eventually to compulsive use. Results from brain region-specific studies lead to a prominent hypothesis that the initial cocaine use is primarily motivated by the nucleus accumbens (NAc)-based reinforcing effects, and transitions to more persistent or habitual drug use by recruiting the dorsal striatum (DS), resulting in escalated cocaine use and resistance to extinction. While the behavioral transition from limited to escalated cocaine use has been observed in both humans and rodent models, the key cocaine-induced cellular adaptations that progress from the NAc to DS to promote this behavioral transition remain underexplored. Targeting this knowledge gap, we focus on the intrinsic membrane excitability (IME) of NAc and DS medium spiny neurons (MSNs). IME determines the ability of neurons to fire action potentials in response to excitatory inputs, and thus directly determines the output of the neurons. Previous results demonstrate a critical IME adaptation?cocaine experience decreases IME of NAc MSNs, and this cocaine-induced IME adaptation in the NAc contributes to psychomotor effects of cocaine, cocaine withdrawal-associated general hypoactive state of the NAc, and cocaine seeking after drug withdrawal. The preliminary results show that during a short-term (5d) cocaine self-administration procedure, mice exhibited limited cocaine taking, and this cocaine procedure only induced the IME adaptation in NAc MSNs, but not DS MSNs. After prolonged (21d) cocaine self-administration, mice exhibited escalated cocaine taking, and the IME adaptation was observed in both NAc and medial/dorsal DS MSNs. Thus, cocaine-induced IME adaptation progresses from the NAc to DS after prolonged cocaine self-administration, correlated to escalated cocaine taking. Furthermore, experimentally preventing cocaine-induced IME adaptation in NAc MSNs prevented the progression of IME adaptation to DS MSNs during prolonged cocaine self-administration, suggesting a critical informational flow from the NAc to DS. This application will explore the anatomical basis mediating the NAc-to- DS progression of cocaine-induced IME adaptation and the behavioral consequence of this progression. The central hypothesis is that the NAc-to-DS progression of cocaine-induced IME adaptation after prolonged cocaine self-administration is mediated, in part, by the striatonigrostriatal ascending spiral, a circuit complex connecting the NAc and DS through reciprocal projections with the ventral tegmental area and substantia nigra, and this NAc-to-DS progression of cocaine-induced IME adaptation promotes the behavioral transition from limited to escalated cocaine use. The proposed experiments will characterize a critical form of cocaine- induced cellular adaptation that progresses from the NAc to DS after prolonged cocaine self-administration. The expected results may provide a circuit mechanism and concrete cellular substrates that mediate the progression of limited drug use toward escalated and eventually compulsive drug use.

Abstract The nucleus accumbens shell (NAcSh) receives glutamatergic projections from several limbic and paralimbic brain regions, with each projection presumably conveying different aspects of emotional and motivational arousals. In motivated behaviors, these excitatory inputs are typically activated concurrently, sending converging excitatory inputs to medium spiny neurons (MSNs), principal neurons in the NAcSh. While recent techniques allow for dissecting individual NAcSh projections, it remains largely unknown whether different NAcSh projections interact with each other when co-activated, and, if so, what the anatomical basis underlies these interactions. To start to address these knowledge gaps, this R21 application focuses on two prominent glutamatergic inputs to the NAcSh, the projections from the basolateral amygdala (BLAp) and paraventricular nucleus of the thalamus (PVTp). Both BLAp and PVTp form monosynaptic contacts onto NAcSh MSNs, but they are differentially involved in NAcSh-based behaviors. With the dual-rhodopsin expression system controlled by tow lasers with different wavelengths, we can simultaneously and independently activate BLAp and PVTp synaptic transmission to the same MSNs. The preliminary results show that a brief co-activation of these two projections induced a short-term potentiation in BLAp transmission but a short-term depression in PVTp transmission. Thus, co-activation temporarily boosted the informational flow through the BLAp while constrained the informational flow through the PCTp. These results not only indicate a clear functional interaction between the BLAp and PVTp, but also provide a potential circuit mechanism through which some motivational arousals override others under certain conditions. The first objective of this application is to extend these preliminary findings by exploring how BLAp and PVTp synapses functionally interact with each other on NAcSh MSNs. Using the dual-color SynapTag technique combined with postsynaptic filling, the second objective of this application is to determine the anatomical arrangement of the BLAp and PVTp that confers the functional interaction of these two projections. Outcomes of the proposed experiments may provide essential functional and anatomical mechanisms through which different aspects of emotional and motivational arousals interact and coordinate for behavioral prioritization.