Andrew L. Eagle

Experience-Dependent Induction of Hippocampal ΔFosB Controls Learning

The hippocampus (HPC) is known to play an important role in learning, a process dependent on synaptic plasticity; however, the molecular mechanisms underlying this are poorly understood. ΔFosB is a transcription factor that is induced throughout the brain by chronic exposure to drugs, stress, and variety of other stimuli and regulates synaptic plasticity and behavior in other brain regions, including the nucleus accumbens. We show here that ΔFosB is also induced in HPC CA1 and DG subfields by spatial learning and novel environmental exposure. The goal of the current study was to examine the role of ΔFosB in hippocampal-dependent learning and memory and the structural plasticity of HPC synapses. Using viral-mediated gene transfer to silence ΔFosB transcriptional activity by expressing ΔJunD (a negative modulator of ΔFosB transcriptional function) or to overexpress ΔFosB, we demonstrate that HPC ΔFosB regulates learning and memory. Specifically, ΔJunD expression in HPC impaired learning and memory on a battery of hippocampal-dependent tasks in mice. Similarly, general ΔFosB overexpression also impaired learning. ΔJunD expression in HPC did not affect anxiety or natural reward, but ΔFosB overexpression induced anxiogenic behaviors, suggesting that ΔFosB may mediate attentional gating in addition to learning. Finally, we found that overexpression of ΔFosB increases immature dendritic spines on CA1 pyramidal cells, whereas ΔJunD reduced the number of immature and mature spine types, indicating that ΔFosB may exert its behavioral effects through modulation of HPC synaptic function. Together, these results suggest collectively that ΔFosB plays a significant role in HPC cellular morphology and HPC-dependent learning and memory. SIGNIFICANCE STATEMENT Consolidation of our explicit memories occurs within the hippocampus, and it is in this brain region that the molecular and cellular processes of learning have been most closely studied. We know that connections between hippocampal neurons are formed, eliminated, enhanced, and weakened during learning, and we know that some stages of this process involve alterations in the transcription of specific genes. However, the specific transcription factors involved in this process are not fully understood. Here, we demonstrate that the transcription factor ΔFosB is induced in the hippocampus by learning, regulates the shape of hippocampal synapses, and is required for memory formation, opening up a host of new possibilities for hippocampal transcriptional regulation.

Role of hippocampal activity-induced transcription in memory consolidation.

Abstract Experience-dependent changes in the strength of connections between neurons in the hippocampus (HPC) are critical for normal learning and memory consolidation, and disruption of this process drives a variety of neurological and psychiatric diseases. Proper HPC function relies upon discrete changes in gene expression driven by transcription factors (TFs) induced by neuronal activity. Here, we describe the induction and function of many of the most well-studied HPC TFs, including cyclic-AMP response element binding protein, serum-response factor, AP-1, and others, and describe their role in the learning process. We also discuss the known target genes of many of these TFs and the purported mechanisms by which they regulate long-term changes in HPC synaptic strength. Moreover, we propose that future research in this field will depend upon unbiased identification of additional gene targets for these activity-dependent TFs and subsequent meta-analyses that identify common genes or pathways regulated by multiple TFs in the HPC during learning or disease.

Single-Prolonged Stress: A Review of Two Decades of Progress in a Rodent Model of Post-traumatic Stress Disorder

Post-traumatic stress disorder (PTSD) is a common, costly, and often debilitating psychiatric condition. However, the biological mechanisms underlying this disease are still largely unknown or poorly understood. Considerable evidence indicates that PTSD results from dysfunction in highly-conserved brain systems involved in stress, anxiety, fear, and reward. Pre-clinical models of traumatic stress exposure are critical in defining the neurobiological mechanisms of PTSD, which will ultimately aid in the development of new treatments for PTSD. Single prolonged stress (SPS) is a pre-clinical model that displays behavioral, molecular, and physiological alterations that recapitulate many of the same alterations observed in PTSD, illustrating its validity and giving it utility as a model for investigating post-traumatic adaptations and pre-trauma risk and protective factors. In this manuscript, we review the present state of research using the SPS model, with the goals of (1) describing the utility of the SPS model as a tool for investigating post-trauma adaptations, (2) relating findings using the SPS model to findings in patients with PTSD, and (3) indicating research gaps and strategies to address them in order to improve our understanding of the pathophysiology of PTSD.

Transcriptional and Epigenetic Regulation of Reward Circuitry in Drug Addiction

Abstract Drug addiction is characterized by compulsive drug seeking and drug intake despite adverse consequences. Although abuse of both licit and illicit substances is estimated to cost over

ΔFosB Decreases Excitability of Dorsal Hippocampal CA1 Neurons

700 billion annually in health care, crime, and lost productivity in the United States alone, there are currently few pharmacological treatments for drug addiction, and those that do exist have low efficacy in many individuals. This paucity of effective treatment options may be due to inextirpable drug-induced changes in brain function that drive some or all of the addiction phenotype. Chronic drug exposure associated with addiction induces persistent changes in the structure and function of brain cells and circuits that underlie addictive behavior, for example, drug seeking and relapse. Therefore, identifying target mechanisms that drive long-term functional changes in the brain is a critical step in illuminating disease etiology and developing new treatments. This will require a comprehensive understanding of the neurobiology of addiction, including the role of gene expression, and its regulation, in drug-induced changes in neuronal structure and function. We will summarize in the subsequent chapter the transcriptional and epigenetic mechanisms of addiction and, furthermore, we will discuss the current implications of these findings in terms of prevention of drug addiction and drug discovery for the treatment of addiction.

GSK3β in the prefrontal cortex: a molecular handle specific to addiction pathology?

Abstract Both the function of hippocampal neurons and hippocampus-dependent behaviors are dependent on changes in gene expression, but the specific mechanisms that regulate gene expression in hippocampus are not yet fully understood. The stable, activity-dependent transcription factor ΔFosB plays a role in various forms of hippocampal-dependent learning and in the structural plasticity of synapses onto CA1 neurons. The authors examined the consequences of viral-mediated overexpression or inhibition of ΔFosB on the function of adult mouse hippocampal CA1 neurons using ex vivo slice whole-cell physiology. We found that the overexpression of ΔFosB decreased the excitability of CA1 pyramidal neurons, while inhibition increased excitability. Interestingly, these manipulations did not affect resting membrane potential or spike frequency adaptation, but ΔFosB overexpression reduced hyperpolarization-activated current. Both ΔFosB overexpression and inhibition decreased spontaneous excitatory postsynaptic currents, while only ΔFosB inhibition affected the AMPA/NMDA ratio, which was mediated by decreased NMDA receptor current, suggesting complex effects on synaptic inputs to CA1 that may be driven by homeostatic cell-autonomous or network-driven adaptations to the changes in CA1 cell excitability. Because ΔFosB is induced in hippocampus by drugs of abuse, stress, or antidepressant treatment, these results suggest that ΔFosB-driven changes in hippocampal cell excitability may be critical for learning and, in maladaptive states, are key drivers of aberrant hippocampal function in diseases such as addiction and depression.

Sex differences in the traumatic stress response: PTSD symptoms in women recapitulated in female rats

The past two decades have seen an explosion of research into the molecular and cellular underpinnings of addiction, with preclinical studies providing evidence that disordered reward processing and decision-making due to altered function of myriad pathways in the medial prefrontal cortex (mPFC) plays a key role in addiction-related behaviors, including alcohol use disorder [1]. However, it is also clear that the neural substrates of various addictions, from drugs to food to sex, are overlapping, and that the pathways that underlie these pathologies are not exclusive to addictive disorders but rather resemble the intersection of circuits that process reward, emotion, memory, and other essential functions [2]. Directly targeting molecular pathways central to the ability of the mPFC to process reward, or to seek rewarding stimuli, in order to treat addiction is thus likely to adversely affect non-pathological behaviors, such as appropriate eating for the drug addict or the normal sex life of the gambling addict. It is therefore critical to uncover the molecular mechanisms unique to the behavioral antecedents of addiction, potentially allowing targeted treatment of alcohol use disorder with reduced untoward effects on the unrelated behaviors of the patient. In the current issue of Neuropsychopharmacology, van der Vaart and colleagues [3] characterize a novel role for mPFC activity of glycogen synthase kinase 3-beta (Gsk3b) in the reinstatement of ethanol seeking—an established model of drug seeking behavior that mirrors drug seeking observed in clinical addiction. Critically, they demonstrate that this role is specific to ethanol relapse, with no effect on relapse to sucrose seeking. The study finds that mPFC GSK3β phosphorylation is increased by acute ethanol exposure, and that AAV-mediated overexpression of GSK3β in mPFC of mice increases ethanol consumption and preference. The authors go on to demonstrate that in rats, operant intake of both ethanol and sucrose is reduced by systemic inhibition of GSK3β, but that subsequent relapse to ethanol seeking after prolonged withdrawal is reduced by systemic GSK3β inhibition while relapse to sucrose seeking is unaffected. Parsing the differential roles of GSK3B in seeking of ethanol vs seeking of a “natural” reward like sucrose can be challenging. The authors found that overexpression of GSK3β in mPFC had an anxiogenic effect during abstinence from ethanol self-administration, suggesting that mPFC GSK3β may play a role in the adverse effects of withdrawal from ethanol. As abstinence from sucrose self-administration does not induce the same negative emotional state found in abstinence from ethanol consumption, it is possible that the specific effects of GSK3β inhibition of relapse to ethanol seeking after prolonged withdrawal are driven by a targeted reduction in the aversive effects of abstinence. It is also possible that these ethanol-specific effects are mediated by the role of GSK3β in ethanol neurotoxicity: GSK3β inhibition provides protection against ethanol neurotoxicity, whereas high GSK3β activity/expression sensitizes neuronal cells to ethanol-induced damage [4]. As ethanol neurotoxicity can contribute to cognitive decline and decreased behavioral inhibition leading to poor decision making, it is possible that reducing this cell loss via GSK3β inhibition could prevent binge drinking and relapse. It is notable that systemic GSK3β inhibition affects the behavioral antecedents of exposure to several drugs of abuse. For instance, GSK3β inhibition increases cocaine selfadministration [5] and interferes with reconsolidation of cocaine-associated reward memories [6]. Although it will be critical to determine whether systemic GSK3β inhibition is effective in preventing relapse to other drugs in a preclinical setting, it is tempting to imagine that targeting this pathway in patients could prevent relapse to several addictive drugs. This could be potentially useful, as most addicts report polysubstance abuse [7]. Finally, the authors suggest that the downstream mediator of mPFC GSK3β effects on ethanol seeking is brain-derived neurotrophic factor (BDNF), as they demonstrate that GSK3β overexpression reduces BDNF message and protein in mPFC. mPFC afferents supply BDNF to the dorsal and ventral striatum, and multiple studies suggest that alcohol increases striatal BDNF as a negative feedback mechanism to suppress alcohol intake [8]. These and other studies have shown that alcohol consumption escalates when this corticostriatal BDNF supply is dysregulated, and thus the work by van der Vaart et al. may provide a novel mechanism whereby ethanol consumption can drive GSK3β-mediated reduction in corticostriatal BDNF secretion to drive relapse to ethanol seeking. As the ubiquitous nature of BDNF in brain function and health makes it a poor therapeutic target, the identification of GSK3β as an upstream mediator of ethanol effects on BDNF and the finding that its systemic inhibition could be used to potentially prevent drug seeking without altering the drive for “natural rewards” (i.e., sucrose) represents a key advance in our understanding and prospective treatment of substance use disorders.

Generation and validation of a floxed FosB mouse line

BackgroundPost-traumatic stress disorder (PTSD) affects men and women differently. Not only are women twice as likely as men to develop PTSD, they experience different symptoms and comorbidities associated with PTSD. Yet the dearth of preclinical research on females leaves a notable gap in understanding the underlying neuropathology of this sex difference.MethodsUsing two standard measures of PTSD-like responses in rats, the acoustic startle response (ASR) and dexamethasone suppression test (DST), we tested the effects of traumatic stress in adult male and female rats using two rodent models of PTSD, single prolonged stress and predator exposure. We then examined the neural correlates underlying these responses with cFos and glucocorticoid receptor immunohistochemistry in brain regions implicated in the traumatic stress response.ResultsWe now report that adult male and female rats across two models of PTSD show consistent sex-specific responses that recapitulate fundamental differences of PTSD in men and women. Trauma-exposed males showed the well-established hyper-responsive phenotype of enhanced ASR and exaggerated negative feedback control of the hypothalamic-pituitary-adrenal axis, while the same traumatic event had little effect on these same measures in females. Dramatic sex differences in how trauma affected cFos and glucocorticoid receptor expression in the brain lend further support to the idea that the trauma response of male and female rats is fundamentally different.ConclusionsTwo standard measures, ASR and DST, might suggest that females are resilient to the effects of traumatic stress, but other measures make it clear that females are not resilient, but simply respond differently to trauma. The next important question to answer is why. We conclude that males and females show fundamentally different responses to trauma that do not simply reflect differences in resilience. The divergent effects of trauma in the brains of males and females begin to shed light on the neurobiological underpinnings of these sex differences, paving the way for improved diagnostics and therapeutics that effectively treat both men and women.

The Role of the Hippocampus in Cocaine Responses

Expression of the FosB gene has been studied extensively in many fields using a variety of tools. However, previous techniques have had a variety of caveats, from potential off-target effects (e.g., overexpression of FosB, ΔFosB, or a dominant negative mutant of JunD, termed ΔJunD) or confounding developmental effects (e.g., the constitutive FosB knockout mouse). Therefore, we sought to create a floxed FosB mouse line that will allow true silencing of the FosB gene with both spatial and temporal control. Here, we detail the cloning strategy, production, and validation of the floxed FosB mouse. We demonstrate methodology for breeding and genotyping, and show that viral-mediated expression of Cre recombinase in a targeted, discrete brain region ablates expression of the FosB gene in floxed but not wild type mice. Thus, the floxed FosB mouse presented here represents an important new tool for the continued investigation of this critical gene.

Differential induction of FosB isoforms throughout the brain by fluoxetine and chronic stress

Experiences generate sensory information that is processed in the cortex then transmitted through the hippocampus, where memories of these experiences are formed, consolidated, and modified. The hippocampus is critical for drug–environment associations, which can underlie relapse to abuse, and control of the mesolimbic dopamine circuitry, which mediates the rewarding effects of the drug. Cocaine regulates hippocampal activity in human addicts and animal models of addiction, and long-term exposure to cocaine changes molecular, structural, and functional properties of hippocampal neurons. Of particular interest are changes in the molecules underlying synaptic plasticity (i.e., long-term potentiation), and associated changes in hippocampal synapse number and strength that accompany exposure to and withdrawal from cocaine. Emerging data indicate that cocaine-driven changes in hippocampal synaptic function, neuronal birth, and projections to other regions may drive key aspects of addiction, and manipulations of these pathways may represent important targets for future therapeutic intervention in cocaine addiction.