Marina E. Wolf, Ph.D. - US grants

Repeated administration results in augmentation of the locomotor stimulant effects of amphetamine (AMPH), a phenomenon known as behavioral sensitization. Dopamine (DA) neurons projecting to the nucleus accumbens (NAc) are thought to play an important role in this process. However, recent studies suggest that AMPH sensitization requires stimulation of N-methyl-D-aspartate (NMDA) receptors since its induction is blocked by MK-801, a noncompetitive NMDA antagonist. Yet, when given alone, MK-801 produces locomotor stimulation and sensitization to this effect occurs after repeated MK-801 administration. The proposed experiments will examine the pharmacological, biochemical and anatomical substrates underlying the ability of NMDA receptor blockade both to prevent AMPH sensitization and to produce sensitization on its own. An important issue is whether MK-801 affects locomotion and sensitization indirectly by altering DA release in the NAc or whether MK-801 acts independently of DA release by blocking NMDA receptors on postsynaptic NAc neurons which may receive convergent DA input. The latter possibility would support the hypothesis that induction of AMPH sensitization involves enhanced glutamatergic transmission, suggesting similarities between sensitization and long - term potentiation. In the proposed studies, in vivo microdialysis will be used to measure extracellular DA and glutamate levels in awake rats while locomotion is measured concurrently using automated photobeam cages. The first Specific Aim is to verify that MK-801 prevents AMPH sensitization by blocking NMDA receptors and to determine whether the NMDA receptors involved in sensitization are located in the NAc. The second Specific Aim is to test the hypothesis that glutamatergic afferents to NAc from hippocampus (HPC) or amygdala (AMY) provide the relevant NMDA receptor stimulation and to examine the possible role of DA projections to HPC and AMY in this neuronal circuit. The third Specific Aim is to determine whether MK-801 blocks AMPH sensitization by interfering with AMPH-induced changes in DA transmission in the NAc, for example, the augmentation of AMPH-stimulated DA release that is generally regarded as a neurochemical correlate of sensitization. The fourth Specific Aim is to test the hypothesis that AMPH sensitization is accompanied by altered glutamate release in the NAc. The fifth Specific Aim is to explore possible mechanistic differences between AMPH and MK-801 sensitization by determining whether behavioral sensitization to MK-801 is accompanied by altered DA or glutamate release in the NAc.

DESCRIPTION (Verbatim from the Applicant's Abstract): Our working hypothesis is that drug addiction is a maladaptive form of glutamate-dependent plasticity. Our prior work has used behavioral sensitization as an animal model for intensification of drug craving. We have shown that glutamate transmission is required for the development of sensitization and that glutamate systems are dramatically altered in sensitized rats. This proposal will further characterize mechanisms underlying changes in glutamate transmission produced by amphetamine or by drug-associated environmental cues. Each of the aims focuses on a different aspect of glutamate's role in sensitization. Aim 1 is based on the hypothesis that the induction of sensitization requires potentiation of AMPA transmission within the ventral tegmental area (VTA). We propose to determine if this is mediated by Narp, a secreted immediate early gene product that clusters AMPA receptors at synaptic sites. Preliminary data show that Narp is elevated in VTA after both acute and repeated amphetamine treatment. Experiments will characterize Narp distribution in VTA, further examine the effects of amphetamine on its expression, and determine if the development of sensitization is prevented by chronic intra-VTA infusion of antisense oligodeoxynucleotides to Narp. Aim 2 builds on prior studies showing that repeated amphetamine decreases GluR1, GluR2 and NR1 levels in the nucleus accumbens (NAc), leading to changes in neuronal excitability that are hypothesized to mediate the long-term maintenance of sensitization. We will compare the persistence of these changes to that of behavioral sensitization, identify cell types within NAc that exhibit altered subunit expression, and examine amphetamine's possible effects on NR2 subunits and NR1 slice variants. Aim 3 will extend our studies of drug-induced plasticity to conditioned locomotion, a phenomenon that may provide insight into situational drug craving. We hypothesize that expression of conditioned locomotion requires increased activity of glutamate projections from prefrontal and cingulate cortices, or amygdala to the NAc. We will determine if the expression of conditioned locomotion is prevented by reversibly inactivating these regions with lidocaine or by blocking AMPA transmission. Microdialysis studies will directly examine the possibility that drug-associated cues activate glutamate transmission. Finally, we will determine if pharmacological treatments that reverse established locomotor sensitization also reverse conditioned locomotion.

DESCRIPTION: (from applicant's abstract) An important new hypothesis for understanding and treating drug addiction is that it may be an inappropriate form of learning. This hypothesis is based on studies demonstrating that glutamate, a transmitter critical for learning and memory, is required for the development of neuroadaptations believed to underlie addiction and relapse. We have shown that the repeated administration of psychostimulants (amphetamine or cocaine) causes profound alterations in glutamate systems. These include alterations in AMPA receptor expression and responsiveness in the nucleus accumbens (Nac), a brain region critical for the rewarding and addictive properties of psychostimulants. Medium spiny GABA-containing neurons, which represent 95% of all neurons in the Nac, receive convergent inputs from midbrain dopamine (DA) neurons and glutamate neurons originating in cortex and limbic regions. Our long-term goal is to understand how psychostimulants, which initially target the DA transporter, ultimately produce adaptations in glutamate transmission in the Nac. This is an important question, because glutamate systems are likely to mediate the neuronal plasticity underlying the transition from drug experimentation to drug dependency. Unfortunately, we know little about chronic interactions between DA and glutamate receptors in the Nac. A logical starting point is to determine how acute DA receptor stimulation can influence glutamate transmission. This application will use postnatal Nac cultures to test the hypothesis that DA receptors modulate the phosphorylation of the AMPA receptor subunit GluR1. We are focusing on AMPA receptors because they are the primary mediators of excitatory transmission in medium spiny neurons and on phosphorylation as a regulatory mechanism because GluR1 phosphorylation leads to marked enhancement of AMPA receptor-mediated currents. Phosphorylation of GluR1 will be detected using phosphorylation site-specific antibodies to GluR1 developed by Dr. Richard Huganir. One selectively recognizes GluR1 phosphorylated on ser-845 [protein kinase A (PKA) site]. The other recognizes GluR1 phosphorylated on ser-831 [protein kinase C (PKC)/Ca2+ -calmodulin dependent protein kinase type II (CaMKII) site]. The first Aim is to characterize conditions regulating basal phosphorylation of GluR1 and the kinases involved in its phosphorylation. First, control cultures will be compared with cultures incubated in the absence of Ca2+, the absence of synaptic activity (TTX, bicuculline, MK-801, CNQX), the absence of inhibitory activity (bicuculline), and the absence of excitatory activity (MK-801, CNQX). Second, we will use activators and inhibitors of PKA, PKC and CaMKII to determine which kinases phosphorylate GluR1 in our cultures. The second Aim is to determine if DA receptor stimulation modulates GluR1 phosphorylation. Experiments will be designed to detect both stimulatory and inhibitory effects of D1 and D2 DA receptor activation. The identity of the residue phosphorylated, combined with experiments involving selective protein kinase inhibitors, will help identify the kinases involved in DA receptor-mediated effects.

My laboratory has been funded by NIDA since 1992 (FIRST Award, 1992-1997; RO1, 1996-2000) to study the role of glutamate in behavioral sensitization to amphetamine. Sensitization is an animal model for the intensification of craving that underlies drug dependency in human addicts. Work supported by my FIRST Award demonstrated that glutamate transmission is required for the development of sensitization and that sensitized rats show dramatic alterations in glutamate neurotransmission. The goal of my R01 is to characterize such alterations and determine how drugs of abuse, which initially target the dopamine (DA) transporter, ultimately produce adaptations in glutamate systems. This is important because it is likely that glutamate mediates the plasticity that underlies the transition from drug experimentation to drug dependency. An understanding of glutamate's role in drug-induced neuroadaptations may lead to the development of pharmacotherapies for the costly and tragic epidemic of addiction. My R01 has the following Aims: 1) To characterize amphetamine's effects on glutamate transmission in the ventral tegmental area (VTA), where sensitization is initiated. 2) To characterize amphetamine's effects on glutamate transmission in the nucleus accumbens (NAc), where sensitization is expressed. 3) To determine if repeated amphetamine administration leads to persistent changes in basal glutamate levels or turnover in VTA or NAc. 4) To determine if amphetamine-induced changes in glutamate transmission in VTA and NAc reflect disinhibition of glutamate-containing projections originating in prefrontal cortex. 5) To characterize effects of repeated amphetamine administration on expression of AMPA and NMDA receptor subunits in these circuits, both at mRNA and protein levels. While studies supported by my R01 have already led to novel hypotheses for glutamate's role in sensitization, we have reached a point where continued progress will require new experimental approaches. I am applying for this K02 Award to obtain relief from administrative and teaching responsibilities that now consume over 60 percent of my time. Under the plan described herein, these responsibilities will be significantly reduced, enabling me to devote 80 percent effort to implementing new approaches and integrating them with my existing research plan. There are 4 specific directions in which I plan to expand our research capabilities: 1) Improving methods for quantifying receptor expression, which is essential for identifying the phenotype(s) of neurons that exhibit amphetamine-induced changes in glutamate receptor expression. 2) Developing primary cultures of NAc neurons as a model system to study dopamine/glutamate receptor interactions. 3) Investigating glutamate involvement in conditioned locomotion, so as to begin to address the crucial role of environmental stimuli in eliciting craving. 4) Developing electrochemical methods for in vivo monitoring of glutamate levels. I have consulted with internationally recognized experts in the development of these new directions, all of whom will continue to provide training and consultation during the period of this Award.

This is a request for extension of Merit Award DA015835. This Award focused on a fundamental question: how do psychomotorstimulants modulate synaptic plasticity at excitatory synapses? To address this, we conducted the first studies of AMPA receptor trafficking, a critical mechanism for regulating synaptic strength, in primary cultures from the nucleus accumbens (NAc) and other reward-related brain regions. We showed that D1 receptors facilitate AMPA receptor synaptic incorporation, providing a mechanism to explain D1 receptor-dependent facilitation of LTP and learning in the normal brain. Abnormal engagement of this mechanism during unregulated DA release may account for maladaptive plasticity during repeated exposure to cocaine. In the next funding period, we will continue to address fundamental questions about plasticity in reward-related brain regions. In particular, we want to understand mechanisms underlying our recent observation that cell surface AMPA receptor levels in the NAc are increased in 2 animal models of addiction: behavioral sensitization and incubation of cocaine craving. Aim 1will determine the subunit composition of AMPA receptor populations in NAc and other addiction-related brain regions. It is critical to know subunit composition, as this determines many features of plasticity. Aim 2 will focus on TARPs (transmembrane AMPA receptor regulatory proteins), a recently discovered family of proteins that regulates AMPA receptor trafficking and function. We will examine TARP localization and function in NAc neurons, the role of TARP phosphorylation in AMPA receptor trafficking, and the role of TARPs in AMPA receptor adaptations that occur in behavioral sensitization and incubation. Aim 3 will focus on synaptic scaling, an important form of homeostatic plasticity in which surface and synaptic expression of AMPA receptors upregulates in response to prolonged hypoactivity of excitatory transmission and downregulates in response to prolonged hyper- activity. We will characterize changes in expression and localization of AMPA receptor subunits and TARPs during synaptic scaling in NAc neurons, as well as TARP phosphorylation. We hypothesize that synaptic scaling, triggered by hypoactivity of cortical inputs during cocaine withdrawal, is responsible for AMPA receptor upregulation in the NAc during sensitization and incubation. Public health relevance: Understanding plasticity mechanisms in addiction will help develop therapies aimed at "unlearning" cues that elicit craving.

Trafficking of glutamate receptors in and out of synapses is an important mechanism for regulating synaptic strength during several types of activity-dependent plasticity, including LTP, LTD and synaptic scaling. Addiction is believed to involve maladaptive neuronal plasticity that rewires motivational circuits, focusing behaviors on drug-seeking. The goal of this study is to use a newly adapted assay to determine whether receptor trafficking contributes to the neuronal plasticity produced by in vivo cocaine exposure, focusing on the nucleus accumbens (NAc) because of its key role in drug reward. Briefly, NAc brain slices are prepared from drug-treated rats and incubated with BS3, a membrane impermeant protein crosslinking agent. BS3 electively crosslinks cell surface receptors, forming high molecular weight aggregates that can be distinguished from intracellular receptors by SDS-PAGE and Western blotting. Using this method, we will irst investigate mechanisms regulating glutamate receptor surface expression in the NAc of naive rats and then characterize changes in glutamate and dopamine (DA) receptor surface expression associatedwith behavioral sensitization to cocaine and cocaine self-administration. In preliminary studies, we found that cocaine-sensitized rats exhibit an increase in AMPA receptor surface expression in the NAc that is correlated with the magnitude of behavioral sensitization. Aim 1will investigate the role of several protein kinases in regulating glutamate receptor surface expression in NAc slices from naive rats: cyclic AMP-dependent protein kinase (PKA), Ca2+/camodulin dependent protein kinase II (CaMKII), and the mitogen-activated protein kinases ERK1/2 and p38 MAPK. They were selected for study because they are important for AMPA receptor trafficking during LTP and also for addiction-related plasticity. In Aim 2, we will determine if acute DA receptor stimulation modulates glutamate receptor trafficking in NAc slices and in vivo, and study the involvement of PKA and ERK1/2 in this modulation. Aim 3 will extend our preliminary studies of cocaine- sensitized rats by examining surface expression of other glutamate receptor subunits and DA receptors, and by evaluating potential correlations between receptor redistribution and activation of signaling pathways. Finally, we will test the hypothesis that intensification ("incubation") of cocaine craving during withdrawal from cocaine self-administration is associated with increased AMPA receptor surface expression in the NAc, enabling more effective activation of NAc neurons by corticolimbic glutamate inputs involved in drug seeking. Relevance to Public Health: Stress or environmental cues associated with cocaine may trigger craving in addicts because their nucleus accumbens neurons are more responsive to the neurotransmitter glutamate. Our study will provide information on how cocaine changes the responsiveness of these neurons. This in turn may help in the design of glutamate-based pharmacological therapies for cocaine craving.

DESCRIPTION (provided by applicant): My lab has played an important role over the past 20 years in showing that neuronal adaptations leading to drug addiction involve glutamate-dependent plasticity. Our recent work is based on evidence that the final common pathway for drug seeking involves cortical and limbic glutamate inputs terminating on nucleus accumbens (NAc) neurons, which in turn project to motor regions responsible for execution of drug seeking. These glutamate inputs activate NAc neurons via AMPA-type glutamate receptors (AMPAR). In light of the importance of AMPAR trafficking for controlling synaptic strength, we are testing the hypothesis that increased trafficking of AMPAR into NAc synapses underlies the enhancement of cocaine seeking that occurs after withdrawal from cocaine exposure. Since 2000, I have been supported by a NIDA K02 Award. Protected time and salary savings afforded by the K02 enabled my lab to develop the innovative methods that led to our current focus on AMPAR trafficking. As a result, we were the first to study AMPAR trafficking in NAc neurons and to establish its importance in rat models of cocaine addiction (behavioral sensitization and incubation of cue-induced cocaine craving after withdrawal from cocaine self-administration). Advances facilitated by the K02 Award led to a NIDA Merit Award and to the continued funding of my R01. These grants focus on defining fundamental properties of AMPAR transmission in the NAc and understanding the mechanisms by which cocaine exposure influences AMPAR trafficking. Recent work funded by these grants has shown that an atypical AMPAR subtype, added to NAc synapses after prolonged withdrawal, mediates enhanced cocaine craving and is therefore a target for the development of anti-craving medications. I am applying for a K05 Award to maintain relief from teaching and service duties so that I can most effectively pursue the Aims of my NIDA grants and position my lab for even stronger contributions to the field through continued innovation. In particular, there are two New Research Directions that I propose to pursue in order to enhance my career development and accelerate our progress towards understanding plasticity mechanisms that contribute to cocaine addiction. First, I want to develop methods to analyze dendritic spine morphology and number, so that I can test the hypothesis that AMPAR synaptic insertion triggers dendritic remodeling in the NAc after cocaine withdrawal. Second, I want to develop the capability to use viral- mediated gene delivery to more effectively explore signaling pathways that underlie cocaine-induced AMPAR, structural and behavioral plasticity. I have identified two expert consultants to assist with the development of these new directions. In addition, protected time resulting from this K05 Award will enable me to expand my contributions as a mentor. This application contains a detailed plan for mentoring young investigators, particularly three Assistant Professors at my institution who I am helping with overall career development as well as the development of NIDA-funded projects. PUBLIC HEALTH RELEVANCE: Learning plays an important role in addiction. For example, environmental cues that addicts have learned to associate with drug availability are powerful triggers for relapse, even long after abstinence is achieved. Our goal is to understand the role of glutamate neurotransmission in addiction-related learning and plasticity, with the goal of identifying targets for the development of anti-craving medications.

There is an urgent need for pharmacological agents that can prevent cue-induced relapse in abstinent cocaine users. In several rat models, cocaine seeking requires glutamate-mediated excitation of medium spiny neurons (MSN) of the nucleus accumbens (NAc). In rats that are drug-naïve or have had limited cocaine exposure, this excitation is mediated primarily by Ca2+-impermeable AMPA receptors (CI-AMPARs). After extended access cocaine self-administration (SA) and withdrawal, cue-induced craving intensifies (?incubates?). We have shown that the expression of incubated cue-induced craving is mediated by Ca2+-permeable AMPARs (CP-AMPARs), which have higher conductance than CI-AMPARs and therefore enable stronger excitation of MSN. The objective of this proposal is to determine if group I metabotropic glutamate receptors (mGluRs), which are expressed postsynaptically in MSN, can be targeted to remove CP-AMPARs from NAc synapses of ?incubated rats?. The central hypothesis is that group I mGluRs normally exert an inhibitory influence on CP-AMPAR levels in NAc synapses, and that decreased group I mGluR signaling during withdrawal permits CP-AMPAR accumulation, whereas stimulating group I mGluRs will remove CP-AMPARs from the NAc of ?incubated rats? and thus reduce craving. This hypothesis, formulated based on preliminary data showing that group I mGluR stimulation removes CP-AMPARs from NAc synapses in slices and cultures, will be tested by pursuing 3 Aims: 1) Determine the effect of group I mGluR activation and blockade on CP-AMPAR levels in NAc MSN in primary culture. Cultured MSN express CP-AMPARs, so this model system can be used to study acute and tonic regulation of CP-AMPAR trafficking by group I mGluRs. 2) Determine the effect of acute group I mGluR activation on AMPAR transmission in the NAc and the expression of cue-induced cocaine seeking. Slice recordings and behavioral studies in ?incubated rats? will determine if acute group I mGluR stimulation removes CP-AMPARs from NAc synapses and reduces craving. 3) Measure group I mGluR surface expression and Homer levels in the NAc after cocaine withdrawal and determine if increasing group I mGluR signaling during withdrawal prevents CP-AMPAR accumulation and incubation. Homers are scaffolding proteins that can mediate group 1 mGluR signaling. Biochemical and behavioral experiments will test the hypothesis that group I mGluR surface expression, levels of long Homer isoforms, and/or mGluR-Homer interactions decrease in the NAc after extended access cocaine SA, permitting accumulation of CP-AMPARs, and that restoring group I mGluR signaling during withdrawal will prevent CP-AMPAR accumulation and incubation of cocaine craving. Our research plan is innovative because it challenges dogma by exploring the therapeutic utility, for addiction, of activating rather than blocking group I mGluRs and because it explores a novel form of synaptic plasticity in the NAc. The significance of our studies is that we expect them to identify a target that can be attacked with existing drugs (group I mGluR positive allosteric modulators) to reduce the risk of relapse during abstinence.

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): Treatments to prevent cue-induced relapse in abstinent cocaine users are urgently needed. In a rat model of persistent relapse vulnerability, extended access cocaine self-administration leads to a withdrawal-dependent incubation of cue-induced cocaine craving. After >1 month of withdrawal, incubation depends on increased synaptic strength in the nucleus accumbens (NAc) mediated by a very persistent elevation of high conductance Ca2+-permeable AMPARs (CP-AMPARs) and by related changes in group I metabotropic glutamate receptor (mGluR) function. Our objective is to determine if maintenance of these long-lasting adaptations depends on dysregulation of protein translation in the NAc. It is well established that dendrites contain mRNAs for AMPAR subunits and other synaptic proteins, and dendritic translation of these mRNAs (controlled by NMDARs and mGluRs) is critical for plasticity. However, nothing is known about whether synaptic transmission regulates translation in the NAc under normal conditions or if drugs of abuse produce persistent alterations in translation in the NAc or in any brain region. By focusing on these fundamental yet unexplored questions, we will advance our understanding of normal NAc function as well as cocaine addiction. Our central hypothesis is: 1) synaptic adaptations in the NAc of incubated rats depend upon dysregulated translation; 2) this dysregulation is due to loss of inhibitory tone on protein translation normally provided by spontaneous NMDAR transmission (minis) and the resulting Ca2+ influx; and 3) loss of this inhibitory tone reflects increased levels of GluN3-containing NMDARs, which have very low Ca2+ permeability. We have 3 Aims. First, we will use metabolic labeling in brain slices to determine if the overall basal rate of protein translatio or the translation of key proteins (GluA1 or Arc) increases after incubation, and if NMDAR and mGluR1/5 regulation of translation is altered. Related questions will be studied in cultured NAc neurons, a system in which dendritic translation can be unequivocally measured. Second, array tomography and postembedding immunogold electron microscopy will be used to define expression and colocalization at the single synapse level of AMPAR subunits, NMDAR subunits, and group I mGluRs. We hypothesize that NAc synapses in incubated rats that are enriched for GluA1 relative to GluA2, indicative of CP-AMPARs, will also be enriched for GluN3. Third, whole-cell patch clamp recordings will determine if incubation is associated with an increased contribution of GluN3-containing NMDARs to synaptic transmission in the NAc, and whether mGluR1 stimulation, which removes CP-AMPARs from synapses, also removes GluN3-NMDARs. Overall, we are testing innovative hypotheses using state-of-the-art methods, several of which have never been applied to addiction research. Furthermore, given that fragile X research has identified aberrant protein translation as a viable therapeutic target, and given intriguing recent evidence that altered protein translation underlies the rapid antidepressant effect of ketamine, our findings will have broad significance for research aimed at normalizing aberrant protein translation in a variety of brain disorders.

In abstinent drug users, environmental cues associated with prior drug use are powerful triggers for relapse. This project focuses on mechanisms that maintain methamphetamine (Meth) craving and vulnerability to relapse even after prolonged periods of abstinence. In a rat model of persistent vulnerability to relapse, cue- induced drug craving progressively intensifies (`incubates') during abstinence from self-administration of many drugs of abuse, including Meth. Despite recent interest in the incubation of Meth craving, studies at the synaptic level are lacking. Such studies have the potential to identify novel therapeutic targets. Recently, we found that strengthening of synapses in the core subregion of the nucleus accumbens (NAc), via incorporation of high conductance Ca2+-permeable AMPARs (CP-AMPARs), underlies incubation of Meth craving after >40 days of abstinence. We also showed that positive allosteric modulators of mGluR1 remove these CP-AMPARs from NAc synapses and thereby reduce `incubated Meth craving'. These findings parallel our previous results on the incubation of cocaine craving. Importantly, however, incubation of craving and CP-AMPAR plasticity occur much more rapidly after discontinuing Meth (1 week) than cocaine (1 month). The narrower time-frame will facilitate identification of underlying synaptic mechanisms. However, first it is necessary to define fundamental parameters of the `incubation model' as it pertains to Meth. The main objective of this proposal is to determine how alterations in ionotropic glutamate receptor transmission in the NAc and its modulation by group I mGluRs contribute to the development and expression of `incubated Meth craving'. Our central hypothesis, formulated based on our cocaine studies and our recently published Meth work, is that incubation- related plasticity in the NAc begins with decreased mGluR1 function, enabling persistent changes in AMPAR and NMDAR transmission, which then maintain incubation of Meth craving. We propose 4 Aims that combine behavioral measures, biochemistry, and whole-cell patch-clamp recordings: 1) Define the time course (rising and falling phases) of incubation of Meth craving and accompanying CP-AMPAR plasticity in female and male rats. 2) Determine the subunit composition of CP-AMPARs that accumulate in the NAc core during incubation of Meth craving and test a potential underlying mechanism. 3) Determine the roles of group I mGluRs in the development and expression of Meth incubation, focusing on mGluR-LTD in the NAc core and on allosteric modulation of group I mGluRs as a potential therapeutic strategy. 4) Determine if NMDAR transmission in the NAc core is altered during the incubation of Meth craving, and if mGluR1 stimulation normalizes NMDAR plasticity. We expect our studies to define fundamental features of the incubation of Meth craving and its relationship to alterations in AMPAR, NMDAR and group I mGluR transmission in the NAc core. This in turn will enable novel research in a number of areas, including sex differences in relapse vulnerability, group I mGluR-based treatment strategies, and consequences of Meth-induced synaptic plasticity for NAc function.

Environmental cues associated with prior cocaine use are powerful triggers for relapse, even in users who have been abstinent for a long period of time. Efforts to understand this persistent vulnerability have revealed that, in rats and humans, cue-induced craving progressively intensifies (`incubates') during abstinence from cocaine self-administration. Our studies of incubation have focused on excitatory synapses in the nucleus accumbens (NAc) core, where GluA2-containing Ca2+-impermeable AMPARs (CI-AMPARs) normally mediate the bulk of AMPAR transmission. We found that strengthening of these synapses, via incorporation of higher conductance Ca2+-permeable AMPARs (CP-AMPARs), occurs after about a month of withdrawal from extended-access cocaine self-administration and thereafter underlies expression of incubated cocaine craving. By understanding the cascade leading to CP-AMPAR accumulation, we may identify novel targets for therapeutic intervention. The goal of this R21 is to test the hypothesis that two direct effectors of cyclic AMP - PKA (protein kinase A) and Epac (exchange protein directly activated by cAMP) - work together to increase the relative contribution of CP-AMPARs to synaptic transmission in the NAc during incubation. Specifically, we hypothesize that, during cocaine withdrawal, Epac activation in NAc medium spiny neurons (MSNs) leads to p38 MAPK activation and removal of CI-AMPARs; this ?makes room? for CP-AMPARs to enter these synapses via a mechanism involving PKA activation. In Aim 1, we will use whole-cell patch-clamp recordings to determine if postsynaptic Epac and p38 MAPK activation leads to removal of CI-AMPARs from NAc synapses in drug-naïve male and female rats, and whether this effect is occluded after incubation of craving. If warranted, we will determine if shRNA knockdown of Epac2 in the NAc core prevents incubation of craving and CP-AMPAR accumulation. Aim 2 will test the effects of postsynaptic PKA activation on AMPAR transmission in MSNs of drug-naïve rats and after incubation of craving, and assess the level of PKA activity in the NAc during incubation. This project is well suited to the R21 mechanism because Epac is a new focus for our lab and the field (a pubmed search on cocaine and Epac revealed only 3 papers) and because very little is known about the role of postsynaptic PKA signaling in regulating AMPAR transmission in NAc MSNs. Significance is high not only because of the need for novel therapeutic approaches to cocaine relapse but because our findings will provide insight into basic mechanisms regulating excitatory synaptic transmission in the NAc, which is relevant to many brain disorders. Finally, this work will set the stage for an R01 application testing the novel hypothesis that Ca2+ signaling in NAc MSNs decreases during cocaine withdrawal; ultimately this disinhibits adenylyl cyclase 5, the predominant isoform in MSNs, leading to Epac/PKA activation and CP-AMPAR insertion.

DESCRIPTION (provided by applicant): Treatments to prevent cue-induced relapse in abstinent cocaine users are urgently needed. In a rat model of persistent relapse vulnerability, extended access cocaine self-administration leads to a withdrawal-dependent incubation of cue-induced cocaine craving. After >1 month of withdrawal, incubation depends on increased synaptic strength in the nucleus accumbens (NAc) mediated by a very persistent elevation of high conductance Ca2+-permeable AMPARs (CP-AMPARs) and by related changes in group I metabotropic glutamate receptor (mGluR) function. Our objective is to determine if maintenance of these long-lasting adaptations depends on dysregulation of protein translation in the NAc. It is well established that dendrites contain mRNAs for AMPAR subunits and other synaptic proteins, and dendritic translation of these mRNAs (controlled by NMDARs and mGluRs) is critical for plasticity. However, nothing is known about whether synaptic transmission regulates translation in the NAc under normal conditions or if drugs of abuse produce persistent alterations in translation in the NAc or in any brain region. By focusing on these fundamental yet unexplored questions, we will advance our understanding of normal NAc function as well as cocaine addiction. Our central hypothesis is: 1) synaptic adaptations in the NAc of incubated rats depend upon dysregulated translation; 2) this dysregulation is due to loss of inhibitory tone on protein translation normally provided by spontaneous NMDAR transmission (minis) and the resulting Ca2+ influx; and 3) loss of this inhibitory tone reflects increased levels of GluN3-containing NMDARs, which have very low Ca2+ permeability. We have 3 Aims. First, we will use metabolic labeling in brain slices to determine if the overall basal rate of protein translatio or the translation of key proteins (GluA1 or Arc) increases after incubation, and if NMDAR and mGluR1/5 regulation of translation is altered. Related questions will be studied in cultured NAc neurons, a system in which dendritic translation can be unequivocally measured. Second, array tomography and postembedding immunogold electron microscopy will be used to define expression and colocalization at the single synapse level of AMPAR subunits, NMDAR subunits, and group I mGluRs. We hypothesize that NAc synapses in incubated rats that are enriched for GluA1 relative to GluA2, indicative of CP-AMPARs, will also be enriched for GluN3. Third, whole-cell patch clamp recordings will determine if incubation is associated with an increased contribution of GluN3-containing NMDARs to synaptic transmission in the NAc, and whether mGluR1 stimulation, which removes CP-AMPARs from synapses, also removes GluN3-NMDARs. Overall, we are testing innovative hypotheses using state-of-the-art methods, several of which have never been applied to addiction research. Furthermore, given that fragile X research has identified aberrant protein translation as a viable therapeutic target, and given intriguing recent evidence that altered protein translation underlies the rapid antidepressant effect of ketamine, our findings will have broad significance for research aimed at normalizing aberrant protein translation in a variety of brain disorders.

Project Summary In this R13 application we request support for the 2nd Gordon Research Conference (GRC) on ?Neurobiology of Drug Addiction,? which will take place in August 2020 in Maine, USA. The objectives of this GRC are to foster open discussion of novel research developments, build new collaborations, and propel the next generation of both scientific advances in addiction research and addiction neuroscience researchers. To achieve these objectives, we will pursue the following four specific aims at the meeting: 1) Provide an international forum to brainstorm about cutting edge, multidisciplinary research at the forefront of addiction neuroscience; 2) Introduce breakthrough neuroscientific techniques that facilitate the understanding of brain mechanisms driving compulsive drug use and relapse; 3) Promote interactions among young and senior investigators and exchange of ideas that will shape the future directions of addiction neuroscience; and 4) Foster development of the next generation of addiction researchers by encouraging the participation of students and postdoctoral fellows who are committed to addiction research. The conference focuses on presentations and discussions at the frontiers of addiction research. The scope of the lectures will be confined to basic and clinical studies involving addictive drugs (including alcohol) and the nervous system and will focus on mechanisms of compulsive drug use and relapse. The meeting will also break down barriers to progress by uniting investigators with synergistic and complementary expertise in areas ranging from molecular mechanisms to translational clinical research and addiction theory. The conference will provide a forum to address the latest developments in addiction research in an open and highly interactive GRC format, which includes formal talks interspersed with ample discussion time, poster sessions, and informal discussion periods that cultivate communication and collaborations. This conference setup differs from that of other meetings in the field and will provide a unique opportunity for close interactions among investigators at all stages of their careers and among investigators with different research approaches and cultural perspectives. Concerted efforts to support participation of students, postdoctoral fellows, young investigators, and under-represented minorities (see budget proposal) will nurture the growth of the next generation of addiction researchers. For 2020, we have added an accompanying GRS to provide a forum for students and postdoctoral fellows; in addition, a substantial number of GRC talks will be delivered by early career PIs. We envision that the Neurobiology of Drug Addiction GRC will significantly advance our understanding of the neural mechanisms underlying drug addiction and stimulate the development of novel hypotheses, research directions, and therapeutic approaches.

Project Summary We will use the `incubation of craving' model to study a major challenge in treating cocaine addiction, that is, the persistence of vulnerability to cue-induced relapse even after long periods of abstinence. In this model, rats exhibit progressive intensification (incubation) of cue-induced craving after withdrawal from extended-access cocaine self-administration. Incubation of craving also occurs in humans. Previously, we showed that Ca2+- permeable AMPARs (CP-AMPARs; homomeric GluA1) accumulate in synapses on medium spiny neurons (MSN) of the nucleus accumbens (NAc) core after ~30 days of withdrawal. Thereafter, their activation is required for expression of incubated cocaine craving. Thus, understanding CP-AMPAR plasticity may provide clues about how the intensity of craving is regulated. Work from our lab and others has shown that cocaine withdrawal is associated with multiple adaptations that predict reduced activity or Ca2+ signaling in NAc MSN. However, little is known about how this might be linked to CP-AMPAR insertion. A promising candidate is a form of homeostatic plasticity involving retinoic acid (RA) signaling. Work in hippocampal neurons has shown that Ca2+ levels associated with ongoing synaptic transmission are sufficient to suppress RA synthesis. However, after a period of inactivity, RA synthesis is disinhibited. This increases GluA1 translation, enabling a homeostatic increase in synaptic strength via insertion of homomeric GluA1 CP-AMPARs. CP-AMPAR accumulation in the NAc core during incubation of craving may likewise involve reduced Ca2+ (see above) and we recently showed that it is associated with increased GluA1 translation. Based on these similarities to RA-mediated homeostatic plasticity in hippocampus and on other data, we hypothesize that cocaine withdrawal is associated with decreased Ca2+ signaling in MSN, leading to disinhibition of RA synthesis, increased GluA1 translation and increased synaptic levels of homomeric GluA1 CP-AMPARs. Aim 1 will test the relationship between excitatory transmission, RA synthesis, and GluA1 translation in cultured NAc MSN (co-cultured with cortical cells to restore glutamate inputs). Aim 2 will use fiber photometry to test the hypothesis that baseline Ca2+ in NAc core MSN is reduced during cocaine withdrawal, although it may transiently increase during cue-induced seeking tests and this increase may be augmented as incubation of craving occurs. Transgenic rats expressing Cre recombinase in D1 or A2a receptor-positive MSN (A2a is a marker for D2 MSN) will enable both MSN populations to be studied. In Aim 3, whole-cell patch clamp recordings in identified D1 or A2a NAc core MSN will determine if acute manipulation of RA signaling alters CP-AMPAR levels and if protein translation is involved. Then, we will determine if knocking down a critical RA synthetic enzyme in NAc core during cocaine withdrawal attenuates CP-AMPAR accumulation and maintenance of incubated craving after long withdrawal. Conversely, we will knock down the enzyme that degrades RA in saline rats to determine if a long-term increase in RA is sufficient to increase CP-AMPARs. Overall, our studies will use state-of-the-art tools to test a conceptually novel hypothesis for incubation of craving.