Shannon L. Gourley - US grants

[unreadable] DESCRIPTION (provided by applicant): The main objective of this project is to characterize the relationship between Extracellular Signal-Regulated Kinase 1/2 (ERK) and cAMP Response Element-Binding Protein (CREB) activity in the extended amygdala and the nucleus accumbens (NAC) core in a long-lasting, stress-related murine model of depression. Although these regions have long been implicated as major substrates for affective, emotional, hedonic, and motivated behavior in humans, little is known about how long-term stress, a major risk factor for depression, disrupts regional activity and connectivity between these and cortical regions to produce depressive-like behaviors consistent with depression in humans. The proposed studies should yield two types of information: First, independent analyses of ERK1/2 and CREB activity (by Western blot) in the sub-regions of the extended amygdala and NAC core will elucidate the role of the signaling pathway in appetitive events that rely on hedonic and motivated processing. A novel, long-lasting model of depression developed and behaviorally characterized in the laboratory will then allow for the investigation of the intracellular mechanisms by which the persistent depressive-like state and antidepressant treatment influence the extended amygdala and NAC core to regulate hedonically-driven and motivated behaviors. We hypothesize the depressive-like phenotype will be characterized by modulated ERK and CREB activity in a regionally-specific manner that co-varies with behavioral outcomes; antidepressant treatment is hypothesized to restore normal ERK/CREB activity. Finally, viral-mediated, local manipulations of CREB are hypothesized to restore motivated responding in depressive animals exposed to prior CORT in a fashion similar to that of antidepressant drugs. Every year, 9.5% of the adult American population will suffer from a depressive illness. Depression carries immense economic, social, and personal costs; however, the manner in which depression impairs mood and motivation is not entirely understood, and contemporary antidepressant drugs are no more effective in treating depression than were first-generation antidepressants developed 50 years ago. Only when the scientific community more fully understands the biological mechanisms of the disease will the medical community be better equipped to rapidly treat depression in patients. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Adolescence is a neurobiologically distinct developmental period characterized by high rates of experimental drug use and vulnerability to the development of substance abuse. Adolescent substance abuse increases the likelihood of developing lifelong addiction, and cocaine addiction emerges with particular virulence-for example, 15-16% of adolescent cocaine users will develop dependence within 10 years of first exposure. Thus, identifying mechanisms of cocaine vulnerability is a critical research imperative. Cocaine and other amphetamine-like psychostimulants potently regulate dendritic spine morphology in the prefrontal cortex. Whether the long-term consequences of cocaine exposure on neural structure are causally related to adolescent vulnerabilities represents a lively debate in field, however direct evidence supporting any single position is limited. This is in part becaus few labs are equipped with the tools to model addiction in animal systems, to capture and enumerate dendritic spine structure, and to manipulate the molecular regulators of dendritic spine structure to isolate causal relationships. We will develop and refine tools by which to identify the impact of cocaine-induced dendritic spine reorganization on decision-making and cocaine vulnerability with the goal of reversing the adverse consequences of early-life cocaine exposure. Throughout, we will focus on ¿1-integrin systems. ¿1-integrin is a receptor for extracellular matrix proteins, and it is implicated in cocaine addiction in humans. Because of widespread expression throughout the CNS, ¿1-integrin is an unrealistic target in developing treatments for addiction; however its downstream effector in cortical neurons, p190RhoGAP, offers a promising target for intervention. p190RhoGAP stabilizes prefrontal cortical cell structure directly by inhibiting actomyosin contraction and indirectly by increasing mRNA expression of Brain-derived neurotrophic factor (Bdnf). Therefore, we propose to develop tools to: 1) inhibit p190RhoGAP function in vivo using viral vector approaches. This presents a significant advance beyond existing tools (the p190rhogap heterozygous mouse), which lack anatomical and temporal selectivity. 2) selectively manipulate the high-affinity BDNF receptor, trkB. This is essential because although BDNF is implicated in addiction etiology, its receptor target in this context remains unknown. This is despite the recent development of a brain-penetrant trkB agonist with therapeutic-like benefits in other mental health domains. 3) pharmacologically reverse the adverse consequences of early-life cocaine exposure. We will develop pharmacological interventions that act on regulators of the actin cytoskeleton (such as BDNF-trkB systems) rather than traditional neurotransmitter targets. We will apply these tools to mice administered cocaine in adolescence with the goal of blocking maladaptive decision-making in adulthood. We will identify correlative relationships between spine structure and behavioral outcomes using high-resolution confocal microscopy, and causal relationships by refining techniques by which to directly manipulate dendritic spine structure in vivo.

DESCRIPTION (provided by applicant): Epidemiological evidence indicates that adolescence represents a period of increased vulnerability to the development of depression, specifically depression that is treatment-resistant. Moreover, treatment options for depressed adolescents are more limited than for adults, and depression onset in adolescence increases the risk of smoking, obesity, substance abuse, unemployment, and depression recurrence across the lifespan. These outcomes may relate to the effects of adversity-such as social isolation or stress hormone exposure- on the prefrontal cortex, which reaches full structural maturity only at the end of adolescence. We and others have hypothesized that the long-term effects of adversity on cellular structure within the prefrontal cortex may be exaggerated when it coincides with the marked neural plasticity of adolescence, and may thereby have additive, persistent, and perhaps even permanent consequences. Empirical evidence is limited, however, because little is known about the behavioral impact of biological events that coordinate structural maturation during adolescence under typical, much less pathological, circumstances. To fill this gap in current knowledge, we will first isolate the neurobiological consequences of early-life adversity on the structure of deep-layer prefrontal cortical neurons. We will utilize in vitro and in vivo imaging, as well as two mouse models of depression that have been developed for male and female adolescents, respectively. This is crucial because adolescent-emergent depression is more common among women, yet female populations remain grossly understudied. Next, to test the potential for therapeutic interventions that target the molecular mechanisms of prefrontal cortical cellular refinement, we will screen two pharmacological compounds that act on regulators of the actin cytoskeleton, measuring their antidepressant-like efficacy. We aim to block the long-term behavioral consequences of early-life adversity. Finally, because depression attenuates reward sensitivity, disrupts decision-making processes essential to accomplishing goals, and diminishes motivation to perform even everyday tasks, we will, as a last aim, use viral-mediated gene silencing and modified surgical disconnection techniques to simultaneously isolate the molecular and neuroanatomical mechanisms of goal-directed action selection. We will focus on molecular interactions critical to postnatal structural refinement: Brain-derived Neurotrophic Factor binding to the high- affinity trkB receptor and formation of the p120RasGAP-p190RhoGAP signaling complex. This proposal is uniquely suited to the NIMH BRAINS program: Using diverse experimental approaches, and drawing on an advisory committee comprised of luminaries in the field, we will chart the trajectory of cellular and behavioral outcomes after early-life adversity; we will refine novel treatment approaches to depression psychopathology in understudied populations; and we will isolate developmental and molecular mechanisms of core components of psychiatric disease.

PROJECT SUMMARY Adolescence is a neurobiologically distinct developmental period characterized by high rates of experimental drug use and vulnerability to the development of substance abuse disorders. Adolescent substance abuse increases the likelihood of developing lifelong addiction, and cocaine addiction emerges with particular virulence-for example, 15-16% of adolescent cocaine users will develop dependence within 10 years of first exposure. Thus, identifying mechanisms of cocaine vulnerability is a critical research imperative. It is now widely accepted that psychostimulant exposure reorganizes dendritic spines within the prefrontal cortex, but whether the long-term consequences of cocaine exposure on neural structure are causally related to addiction vulnerability at any age represents a lively debate in the field. This is in part because few labs are equipped with the tools to model addiction in animal systems, to capture and enumerate dendritic spines, and to manipulate the molecular regulators of dendritic spine structure in discrete neurocircuits in order to isolate causal relationships. We will apply precisely these tools to identify the organizational and behavioral impact of adolescent cocaine self-administration, with the ultimate goal of reversing the adverse consequences of early- life cocaine exposure. As a model system, we use mice, which like humans, readily self-administer cocaine. We will focus on orbitofrontal cortical Brain-derived Neurotrophic Factor (BDNF) and its high-affinity receptor trkB. Postnatal BDNF expression is a critical determinant of adolescent cortical spine development and refinement. However, early-life stimulant exposure decreases Bdnf in the orbitofrontal cortex, a structure widely implicated in addiction pathology. Thus, BDNF systems may present a promising target in reversing the organizational and functional consequences of adolescent cocaine self-administration. We propose 3 discrete experiments using experimental protocols already established in my lab: 1) We will isolate and reconstruct in 3D deep-layer orbitofrontal cortical neurons from mice that had self- administered cocaine in adolescence and then showed either behavioral vulnerability or resilience to cocaine seeking and stimulus-response habit formation in adulthood. We hypothesize that cocaine vulnerability will be associated with neural simplification in deep-layer orbitofrontal cortex. 2) We will block the long-term behavioral effects of adolescent cocaine self-administration (context-induced cocaine seeking and stimulus-response habit formation) with neurotrophin-based intervention strategies. Specifically, we expect that treating cocaine-exposed adolescent mice with the novel trkB agonist 7,8-DHF will occlude the long-term negative impact of early-life cocaine self-administration. 3) Finally, we will test a neuroanatomical model in which orbitofrontal cortical Bdnf deficiency results in stimulus-response habits due to perturbations in an orbitofrontal-amygdala neurocircuit. Substantial preliminary findings support each aim and will ensure the completion of this project.

PROJECT SUMMARY Adolescent cocaine abuse increases the risk and severity of lifelong addiction and decreases the likelihood that cocaine-abusing individuals will seek treatment. Developing and understanding therapeutic approaches that mitigate maladaptive decision-making and cocaine-seeking behaviors in organisms with a history of cocaine exposure during adolescence could reduce the high societal cost of cocaine addiction. During adolescence, dendritic spines in the prefrontal cortex (PFC), a brain region critical for complex decision-making, proliferate markedly and then prune, refine, and mature. This process is believed to optimize cellular connectivity and set the neural stage for adult functioning, but such dramatic cellular reorganization within a narrow developmental period may also open a window of vulnerability to insults. We have shown that in adolescent mice, experimenter-administered cocaine derails neural development in the orbital PFC (oPFC), eliminating dendrites and dendritic spines. It also impairs complex decision-making, accelerating the development of stimulus-response habits in adulthood. We have also shown that adolescent mice develop marked individual differences in their cocaine self-administration patterns, and that mice self- administering escalating amounts of cocaine most readily develop stimulus-response habits in adulthood. In Aim I, we will test the hypothesis that individual differences in cocaine self-administration in adolescence determine effects on oPFC dendritic spines, such that mice that escalate are more susceptible to spine deficiencies in adulthood. We will then assess whether ifenprodil, an NR2B-selective NMDA receptor antagonist that blocks the reinstatement of heroin-, nicotine-, and alcohol-seeking behaviors in rodent models will also have therapeutic-like effects after adolescent cocaine exposure, occluding cocaine-induced habits. In humans, adolescent cocaine exposure increases the risk of substance use, dependence, and relapse in adulthood. In Aim II, we will examine whether individual differences in cocaine self-administration in adolescence are associated with individual differences in cocaine self-administration and the reinstatement of cocaine seeking in adulthood. We expect that mice with a history of escalating cocaine exposure will respond more for cocaine as adults and be more likely to reinstate responding after extinction conditioning. In these experiments, ifenprodil will be paired with extinction training in an attempt to mitigate the reinstatement of cocaine seeking. This approach models the use of ifenprodil as a therapeutic adjunct to behavioral therapy in humans and is strongly supported by our preliminary findings. Finally, we will test the utility of ifenprodil as an adolescent-targeted intervention strategy. Cortical development is characterized by a reduction in NMDA NR2B:NR2A subunit ratios, which facilitates spine and synapse stabilization. We hypothesize that application of the NR2B-selective antagonist ifenprodil during critical adolescent developmental periods will have corrective behavioral benefits that persist into adulthood.

SUMMARY PI3-kinase (PI3K) is a membrane-associated signaling complex that phosphorylates phosphoinositides, second messengers that regulate neuronal development, survival, and plasticity. In 2002, Izzo et al. reported that intraventricular PI3K inhibition blocks the expression of cocaine-induced psychomotor sensitization (Nature Neurosci.). Subsequent studies indicated that repeated cocaine exposure can increase PI3K activity in the medial prefrontal cortex (mPFC). Nevertheless, causal relationships between mPFC PI3K and cocaine- induced behavioral sequelae remain unconfirmed. We will directly manipulate the PI3K subunit p110? to mitigate stimulus-elicited habits following cocaine. p110? is one of the four PI3K catalytic subunits, and it is highly expressed throughout postnatal development and in adulthood. We find that reduction of Pik3cb, encoding p110?, broadly throughout the mPFC blocks stimulus-elicited habits and locomotor sensitization following cocaine exposure during adolescence or young adulthood. These are periods of considerable drug experimentation in humans. In Aim 1, we will use viral-mediated gene silencing to identify specific mPFC subregions responsible for the ?protective? consequences of Pik3cb inhibition. One widely-reported consequence of repeated cocaine exposure is an imbalance in dopamine receptor- mediated signaling, favoring D1-family Gs-coupled, at the expense of D2-family Gi-coupled, systems. D1 stimulation activates PI3K and is a likely mechanism by which psychostimulants strengthen habit-based behavior. Overexpression of Drd1 in the mPFC decreases D2 expression in the downstream striatum, suggesting that normalization of mPFC D1-mediated signaling following cocaine could engage striatal D2 systems. In Aim 2, we will test the hypothesis that Pik3cb inhibition in the mPFC creates a permissive environment for dorsomedial striatal D2-dependent goal-directed response strategies. Cocaine can induce activity-dependent dendritic spine proliferation in the mPFC. Meanwhile, inhibiting PI3K p110? can normalize aberrant dendritic spine proliferation in models of Fragile X Syndrome. Thus, inhibiting p110? could conceivably correct cocaine-induced spinogenesis. Further, PI3K p110? and ? regulate RhoA GTPase-dependent neuronal contraction and NMDA receptor-dependent long-term depression, respectively. In Aim 3, we will test the hypothesis that inhibiting p110? and ? will correct dendritic spine densities and block habits following cocaine, while p110? inhibition could exacerbate cocaine?s influence. Our findings indicate that p110? blockade confers certain behavioral resiliencies to cocaine. The proposed studies will crystallize anatomical mechanisms and cyto-structural consequences. Knowledge gained from these experiments could advance treatment strategies for drug use disorders, given that subunit-selective inhibitors may have more favorable clinical profiles than broad-spectrum PI3K blockade.

Elevated glucocorticoids, particularly during specific developmental periods, cause long-term biases towards habit-based behaviors that are linked with depression, obesity, and other maladaptive outcomes in adulthood. Neurobiological mechanisms remain largely unclear. Integrin receptors are cell adhesion factors linked with the stress response system and genetic risk for neurodevelopmental disease. Composed of an ? subunit responsible for ligand binding and a ? subunit that activates intracellular signaling, integrins respond to extracellular matrix proteins, influencing cell structure through downstream cytoskeletal signaling factors. Integrin-mediated signaling stabilizes cell structure in the transition from adolescence to adulthood, such that genetic ablation of the ?1 subunit, highly expressed in the cortex and hippocampus, causes dendritic spine loss starting in adolescence. In humans, ITGB1, encoding ?1-integrin, is identified in genome-wide association studies of depression and schizophrenia, diseases characterized by deficits in PFC-dependent planning and action. Despite connections with neurodevelopmental disease, ?1-integrin involvement in PFC-dependent action selection remains opaque. We will test the hypothesis that a ?1-integrin-Abl2/Arg-cortactin-ROCK2 signaling axis coordinates goal-directed action selection and thus, is a sensible target for blocking habits due to glucocorticoid and stressor exposure. Aligned with RDoC-defined positive valence domains, specific aims are: Aim 1. To identify how the ?1-integrin-Arg-cortactin-ROCK2 signaling axis influences oPFC- dependent action selection. We will use a combination of viral-mediated gene silencing and pharmacological manipulations to test the hypothesis that ?1-integrin-Arg-cortactin-ROCK2 interactions in the oPFC coordinate goal-directed response choice, countering inflexible habits. Next, we will test the hypothesis that ?1-integrin- dependent oPFC interactions with the basolateral amygdala support goal-directed response choice. Last, we will test the hypothesis that site-selective Itgb1 silencing structurally phenocopies glucocorticoid exposure, eliminating dendritic spines on excitatory neurons within the oPFC. Aim 2. To mitigate stressor-related habits and dendritic spine abnormalities in the oPFC. Next, we will test the hypothesis that stimulation of Arg and cortactin will block habits and changes in dendritic spine densities and morphologies following developmental corticosterone or exposure to social isolation. This aim will reveal strategies by which to correct cyto-structural change and habit biases following adversity. Aim 3. To reveal functional interactions with tyrosine receptor kinase B (trkB). Activation of ?1-integrin- mediated signaling events that inhibit ROCK2 stimulates BDNF, which binds to its high-affinity receptor trkB. ROCK2 inhibition also modifies the ratio of full-length/truncated trkB in the PFC, favoring the active full-length isoform, and it enhances action-outcome memory in multiple contexts. Our final aim will test the hypothesis that the enrichment of action-outcome decision making (blocking habits) via ROCK2 inhibition is trkB-dependent.

Project Summary (Project 3, Rainnie) The ability to recognize the identity and intentions of others and to react accordingly is an evolutionarily adaptive process with relevance to psychiatric disorders. However, the cellular and neurotransmitter systems that regulate this process remain largely unknown. One region consistently shown to play a pivotal role in regulating the behavioral response to both appetitive and aversive sensory and/or social stimuli is the basolateral amygdala (BLA). The activity of neurons in the BLA has been shown to signal preference in a social recognition task, and in the previous Conte Center funding period we showed that during social interaction between same sex rats neural activity in the BLA and a key component of reward circuitry, the nucleus accumbens (NAc), became highly synchronized. Synchronization was associated with markedly enhanced ?-? cross-frequency-coupling (?-? CFC) similar to that seen in non-human primates (NHP) during performance of a social preference task (see Project 4) and between the mPFC and NAc during pair bonding in voles (Project 2). Together, these data suggest that ?-? CFC may represent a canonical mechanism for integrating executive, emotion, and reward circuits to drive appropriate behavioral responses during social interaction. We have successfully developed a novel rat social recognition task, which mirrors the task being utilized in the NHP studies of Project 4, and with which we can directly examine the role of two neurotransmitters, acetylcholine (ACh) and oxytocin (OT) in the modulation of social recognition as well as ?-? CFC in the pathway from the BLA to the NAc. These two neurotransmitters have been shown to play key roles in regulating cue discrimination, ?-? CFC, and social interaction in rodents and NHPs. However, ACh and OT are usually studied independently of one another. It is our contention that ACh and OT act synergistically in the BLA to facilitate social recognition in conspecifics. Here, we will test the hypothesis that OT release in the BLA acts to facilitate social recognition by enhancing ACh release in the BLA and promoting ?-? CFC in the pathway from the BLA to NAc. To challenge this hypothesis we will use state-of- the-art gene transfer and gene deletion techniques in conjunction with pathway specific viral vector manipulations to selectively target specific neural circuits that are thought to regulate BLA neural activity during social recognition and discrimination. The PI of Project 3, is an internationally recognized expert in the field of amygdala anatomy and physiology and has a track record of using state-of-the-art viral vector manipulations to examine the fine structure of neural circuits that regulate affective behavior. In addition, the research team for Project 3 have all of the necessary expertise to successfully complete the proposed studies. We anticipate that at the end of Project 3 we will have markedly increased our understanding of the interaction between two critical neurotransmitter systems that are known to play a major role in social discrimination. By better understanding the systems and circuits that guide prosocial behavior we will be able to develop more targeted therapeutic approaches for disorders that share a common pathology of deficits in social behavior.