A dual-pathway architecture enables chronic stress to promote habit formation.

Chronic stress can change how we learn and, thus, how we make decisions by promoting the formation of inflexible, potentially maladaptive, habits. Here we investigated the neuronal circuit mechanisms that enable this. Using a multifaceted approach in male and female mice, we reveal a dual pathway, amygdala-striatal, neuronal circuit architecture by which a recent history of chronic stress shapes learning to disrupt flexible goal-directed behavior in favor of inflexible habits. Chronic stress inhibits activity of basolateral amygdala projections to the dorsomedial striatum to impede the action-outcome learning that supports flexible, goal-directed decisions. Stress also increases activity in direct central amygdala projections to the dorsomedial striatum to promote the formation of rigid, inflexible habits. Thus, stress exerts opposing effects on two amygdala-striatal pathways to promote premature habit formation. These data provide neuronal circuit insights into how chronic stress shapes learning and decision making, and help understand how stress can lead to the disrupted decision making and pathological habits that characterize substance use disorders and other psychiatric conditions.

Neural substrates of habit.

Active reward pursuit is supported by the balance between the cognitive and habitual control of behavior. The cognitive, goal-directed strategy relies on the prospective evaluation of anticipated consequences, which allows behavior to readily adapt when circumstances change. Repetition of successful actions promotes less cognitively taxing habits, in which behavior is automatically executed without prospective consideration. Disruption in either of these behavioral regulatory systems contributes to the symptoms that underlie many psychiatric disorders. Here, I review recently identified neural substrates, at multiple neural levels, that contribute to habits and outline gaps in knowledge that must be addressed to fully understand the neural mechanisms of behavioral control.

Distinct cortical-amygdala projections drive reward value encoding and retrieval.

The value of an anticipated rewarding event is a crucial component of the decision to engage in its pursuit. But little is known of the networks responsible for encoding and retrieving this value. By using biosensors and pharmacological manipulations, we found that basolateral amygdala (BLA) glutamatergic activity tracks and mediates encoding and retrieval of the state-dependent incentive value of a palatable food reward. Projection-specific, bidirectional chemogenetic and optogenetic manipulations revealed that the orbitofrontal cortex (OFC) supports the BLA in these processes. Critically, the function of ventrolateral and medial OFC→BLA projections is doubly dissociable. Whereas lateral OFC→BLA projections are necessary and sufficient for encoding of the positive value of a reward, medial OFC→BLA projections are necessary and sufficient for retrieving this value from memory. These data reveal a new circuit for adaptive reward valuation and pursuit and provide insight into the dysfunction in these processes that characterizes myriad psychiatric diseases.

Regulation of habit formation in the dorsal striatum.

Habits are an essential and pervasive component of our daily lives that allow us to efficiently perform routine tasks. But their disruption contributes to the symptoms that underlie many psychiatric diseases. Emerging data are revealing the cellular and molecular mechanisms of habit formation in the dorsal striatum. New data suggest that in both the dorsolateral and dorsomedial striatum histone deacetylase (HDAC) activity acts as a critical negative regulator of the transcriptional processes underlying habit formation. In this review, we discuss this recent work and draw conclusions relevant to the treatment of diseases marked by maladaptive habits.

Habits Are Negatively Regulated by Histone Deacetylase 3 in the Dorsal Striatum.

BACKGROUND: Optimal behavior and decision making result from a balance of control between two strategies, one cognitive/goal-directed and one habitual. These systems are known to rely on the anatomically distinct dorsomedial and dorsolateral striatum, respectively. However, the transcriptional regulatory mechanisms required to learn and transition between these strategies are unknown. Here we examined the role of one chromatin-based transcriptional regulator, histone modification via histone deacetylases (HDACs), in this process. METHODS: We combined procedures that diagnose behavioral strategy in rats with pharmacological and viral-mediated HDAC manipulations, chromatin immunoprecipitation, and messenger RNA quantification. RESULTS: The results indicate that dorsal striatal HDAC3 activity constrains habit formation. Systemic HDAC inhibition following instrumental (lever press → reward) conditioning increased histone acetylation throughout the dorsal striatum and accelerated habitual control of behavior. HDAC3 was removed from the promoters of key learning-related genes in the dorsal striatum as habits formed with overtraining and with posttraining HDAC inhibition. Decreasing HDAC3 function, either by selective pharmacological inhibition or by expression of dominant-negative mutated HDAC3, in either the dorsolateral striatum or the dorsomedial striatum accelerated habit formation, while HDAC3 overexpression in either region prevented habit. CONCLUSIONS: These results challenge the strict dissociation between dorsomedial striatum and dorsolateral striatum function in goal-directed versus habitual behavioral control and identify dorsostriatal HDAC3 as a critical molecular directive of the transition to habit. Because this transition is disrupted in many neurodegenerative and psychiatric diseases, these data suggest a potential molecular mechanism for the negative behavioral symptoms of these conditions and a target for therapeutic intervention.

Basolateral amygdala rapid glutamate release encodes an outcome-specific representation vital for reward-predictive cues to selectively invigorate reward-seeking actions.

Environmental stimuli have the ability to generate specific representations of the rewards they predict and in so doing alter the selection and performance of reward-seeking actions. The basolateral amygdala participates in this process, but precisely how is unknown. To rectify this, we monitored, in near-real time, basolateral amygdala glutamate concentration changes during a test of the ability of reward-predictive cues to influence reward-seeking actions (Pavlovian-instrumental transfer). Glutamate concentration was found to be transiently elevated around instrumental reward seeking. During the Pavlovian-instrumental transfer test these glutamate transients were time-locked to and correlated with only those actions invigorated by outcome-specific motivational information provided by the reward-predictive stimulus (i.e., actions earning the same specific outcome as predicted by the presented CS). In addition, basolateral amygdala AMPA, but not NMDA glutamate receptor inactivation abolished the selective excitatory influence of reward-predictive cues over reward seeking. These data support [corrected] the hypothesis that transient glutamate release in the BLA can encode the outcome-specific motivational information provided by reward-predictive stimuli.

Early postnatal nicotine exposure causes hippocampus-dependent memory impairments in adolescent mice: Association with altered nicotinic cholinergic modulation of LTP, but not impaired LTP.

Fetal nicotine exposure from smoking during pregnancy causes long-lasting cognitive impairments in offspring, yet little is known about the mechanisms that underlie this effect. Here we demonstrate that early postnatal exposure of mouse pups to nicotine via maternal milk impairs long-term, but not short-term, hippocampus-dependent memory during adolescence. At the Schaffer collateral (SC) pathway, the most widely studied synapses for a cellular correlate of hippocampus-dependent memory, the induction of N-methyl-D-aspartate receptor-dependent transient long-term potentiation (LTP) and protein synthesis-dependent long-lasting LTP are not diminished by nicotine exposure, but rather unexpectedly the threshold for LTP induction becomes lower after nicotine treatment. Using voltage sensitive dye to visualize hippocampal activity, we found that early postnatal nicotine exposure also results in enhanced CA1 depolarization and hyperpolarization after SC stimulation. Furthermore, we show that postnatal nicotine exposure induces pervasive changes to the nicotinic modulation of CA1 activity: activation of nicotinic receptors no longer increases CA1 network depolarization, acute nicotine inhibits rather than facilitates the induction of LTP at the SC pathway by recruiting an additional nicotinic receptor subtype, and acute nicotine no longer blocks LTP induction at the temporoammonic pathway. These findings reflect the pervasive impact of nicotine exposure during hippocampal development, and demonstrate an association of hippocampal memory impairments with altered nicotinic cholinergic modulation of LTP, but not impaired LTP. The implication of our results is that nicotinic cholinergic-dependent plasticity is required for long-term memory formation and that postnatal nicotine exposure disrupts this form of plasticity.

Exercise and sodium butyrate transform a subthreshold learning event into long-term memory via a brain-derived neurotrophic factor-dependent mechanism.

We demonstrate that exercise enables hippocampal-dependent learning in conditions that are normally subthreshold for encoding and memory formation, and depends on hippocampal induction of brain-derived neurotrophic factor (BDNF) as a key mechanism. Using a weak training paradigm in an object location memory (OLM) task, we show that sedentary mice are unable to discriminate 24 h later between familiar and novel object locations. In contrast, 3 weeks of prior voluntary exercise enables strong discrimination in the spatial memory task. Cognitive benefits of exercise match those attained with post-training sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor previously shown to enable subthreshold learning. We demonstrate that the enabling effects of exercise and NaB on subthreshold OLM learning are dependent on hippocampal BDNF upregulation, and are blocked by hippocampal infusion of BDNF short-interfering RNA. Exercise and NaB increased bdnf transcripts I and IV, and the increases were associated with BDNF promoter acetylation on H4K8 but not H4K12. These data provide support for the concept that exercise engages epigenetic control mechanisms and serves as a natural stimulus that operates in part like NaB and potentially other HDAC inhibitors, placing the brain into a state of readiness for plasticity.

HDAC3-selective inhibitor enhances extinction of cocaine-seeking behavior in a persistent manner.

Nonspecific histone deacetylase (HDAC) inhibition has been shown to facilitate the extinction of drug-seeking behavior in a manner resistant to reinstatement. A key open question is which specific HDAC is involved in the extinction of drug-seeking behavior. Using the selective HDAC3 inhibitor RGFP966, we investigated the role of HDAC3 in extinction and found that systemic treatment with RGFP966 facilitates extinction in mice in a manner resistant to reinstatement. We also investigated whether the facilitated extinction is related to the enhancement of extinction consolidation during extinction learning or to negative effects on performance or reconsolidation. These are key distinctions with regard to any compound being used to modulate extinction, because a more rapid decrease in a defined behavior is interpreted as facilitated extinction. Using an innovative combination of behavioral paradigms, we found that a single treatment of RGFP966 enhances extinction of a previously established cocaine-conditioned place preference, while simultaneously enhancing long-term object-location memory within subjects. During extinction consolidation, HDAC3 inhibition promotes a distinct pattern of histone acetylation linked to gene expression within the infralimbic cortex, hippocampus, and nucleus accumbens. Thus, the facilitated extinction of drug-seeking cannot be explained by adverse effects on performance. These results demonstrate that HDAC3 inhibition enhances the memory processes involved in extinction of drug-seeking behavior.

Differential roles for Nr4a1 and Nr4a2 in object location vs. object recognition long-term memory.

Nr4a1 and Nr4a2 are transcription factors and immediate early genes belonging to the nuclear receptor Nr4a family. In this study, we examine their role in long-term memory formation for object location and object recognition. Using siRNA to block expression of either Nr4a1 or Nr4a2, we found that Nr4a2 is necessary for both long-term memory for object location and object recognition. In contrast, Nr4a1 appears to be necessary only for object location. Indeed, their roles in these different types of long-term memory may be dependent on their expression in the brain, as NR4A2 was found to be expressed in hippocampal neurons (associated with object location memory) as well as in the insular and perirhinal cortex (associated with object recognition memory), whereas NR4A1 showed minimal neuronal expression in these cortical areas. These results begin to elucidate how NR4A1 and NR4A2 differentially contribute to object location versus object recognition memory.

Brain-penetrant LSD1 inhibitors can block memory consolidation.

Modulation of histone modifications in the brain may represent a new mechanism for brain disorder therapy. Post-translational modifications of histones regulate gene expression, affecting major cellular processes such as proliferation, differentiation, and function. An important enzyme involved in one of these histone modifications is lysine specific demethylase 1 (LSD1). This enzyme is flavin-dependent and exhibits homology to amine oxidases. Parnate (2-phenylcyclopropylamine (2-PCPA); tranylcypromine) is a potent inhibitor of monoamine oxidases and derivatives of 2-PCPA have been used for development of selective LSD1 inhibitors based on the ability to form covalent adducts with flavin adenine dinucleotide (FAD). Here we report the synthesis and in vitro characterization of LSD1 inhibitors that bond covalently to FAD. The two most potent and selective inhibitors were used to demonstrate brain penetration when administered systemically to rodents. First, radiosynthesis of a positron-emitting analog was used to obtain preliminary bio-distribution data and whole brain time-activity curves. Second, we demonstrate that this series of LSD1 inhibitors is capable of producing a cognitive effect in a mouse model. By using a memory formation paradigm, novel object recognition, we show that LSD1 inhibition can abolish long-term memory formation without affecting short-term memory, providing further evidence for the importance of reversible histone methylation in the function of the nervous system.

CBP in the nucleus accumbens regulates cocaine-induced histone acetylation and is critical for cocaine-associated behaviors.

Cocaine exposure triggers molecular events that lead to long-lasting changes in brain structure and function. These changes can lead to the development of persistent and robust behavioral adaptations that characterize addiction. Recent evidence suggests the regulation of transcription via chromatin modification, such as histone acetylation, has an important role in the development of addictive behavior. Histone acetylation is regulated by histone acetyltransferases (HATs), which acetylate histones and promote transcription, and histone deacetylases (HDACs), which remove acetyl groups and silence transcription. Studies have demonstrated that HDACs may negatively regulate cocaine-induced behaviors, but very little is known about the role of specific HATs in long-lasting drug-induced plasticity. The histone acetyltransferase CREB-binding protein (CBP) mediates transcriptional activation by recruiting basal transcription machinery and acetylating histones. CBP is a critically important chromatin-modifying enzyme involved in regulating gene expression required for long-term plasticity and memory. However, the role of CBP in cocaine-induced behaviors remains largely unknown. We examined the role of CBP in drug-induced plasticity using CBP-FLOX genetically modified mice in combination with adeno-associated virus expressing Cre-recombinase to generate focal homozygous deletions of Cbp in the nucleus accumbens (NAc). A complete loss of CBP in NAc neurons results in decreased histone acetylation and significantly altered c-fos expression in response to cocaine. Furthermore, the deletion of CBP in the NAc correlates with significant impairments in cocaine sensitivity and context-cocaine associated memory. This is the first study to demonstrate a definitive role for CBP in modulating gene expression that may subserve drug-seeking behaviors.

Hippocampal focal knockout of CBP affects specific histone modifications, long-term potentiation, and long-term memory.

To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBP's homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories.

Membrane-associated glucocorticoid activity is necessary for modulation of long-term memory via chromatin modification.

Glucocorticoid hormones enhance the consolidation of long-term memory of emotionally arousing training experiences. This memory enhancement requires activation of the cAMP-dependent kinase pathway and the subsequent phosphorylation of cAMP response-element binding (CREB) protein. Here, we demonstrate that glucocorticoids enhance the consolidation of hippocampus-dependent and hippocampus-independent aspects of object recognition memory via chromatin modification. More specifically, systemic corticosterone increases histone acetylation, a form of chromatin modification, in both the hippocampus and insular cortex following training on an object recognition task. This led us to examine whether increasing histone acetylation via histone deacetylase (HDAC) inhibition enhances memory in a manner similar to corticosterone. We found a double dissociation between posttraining HDAC inhibitor infusion into the insular cortex and hippocampus on the enhancement of object recognition and object location memory, respectively. In determining the molecular pathway upstream of glucocorticoids' effects on chromatin modification, we found that activation of membrane-associated glucocorticoid receptors (GRs) and the subsequent interaction between phospho-CREB and CREB-binding protein (CBP) appear to be necessary for glucocorticoids to enhance memory consolidation via chromatin modification. In contrast, mineralocorticoid receptors (MRs) do not appear to be involved. The findings also indicate that glucocorticoid activity has differential influences on hippocampus-dependent and hippocampus-independent components of memory for objects.

Reversal-specific learning impairments after a binge regimen of methamphetamine in rats: possible involvement of striatal dopamine.

A growing body of evidence indicates that protracted use of methamphetamine (mAMPH) causes long-term impairments in cognitive function in humans. Aside from the widely reported problems with attention, mAMPH users exhibit learning and memory deficits, particularly on tasks requiring response control. Although binge mAMPH administration to animals results in cognitive deficits, few studies have attempted to test behavioral flexibility in animals after mAMPH exposure. The aim of this study was to evaluate whether mAMPH would produce impairments in two tasks assessing flexible responding in rats: a touchscreen-based discrimination-reversal learning task and an attentional set shift task (ASST) based on a hallmark test of executive function in humans, the Wisconsin Card Sort. We treated male Long-Evans rats with a regimen of four injections of 2 mg/kg mAMPH (or vehicle) within a single day, a dosing regimen shown earlier to produce object recognition impairments. We then tested them on (1) reversal learning after pretreatment discrimination learning or (2) the ASST. Early reversal learning accuracy was impaired in mAMPH-treated rats. MAMPH pretreatment also selectively impaired reversal performance during ASST testing, leaving set-shifting performance intact. Postmortem analysis of [(125)I]RTI-55 binding revealed small (10-20%) but significant reductions in striatal dopamine transporters produced by this mAMPH regimen. Together, these results lend new information to the growing field documenting impaired cognition after mAMPH exposure, and constitute a rat model of the widely reported decision-making deficits resulting from mAMPH abuse seen in humans.

Modulation of chromatin modification facilitates extinction of cocaine-induced conditioned place preference.

BACKGROUND: Recent evidence suggests that epigenetic mechanisms have an important role in the development of addictive behavior. However, little is known about the role of epigenetic mechanisms in the extinction of drug-induced behavioral changes. In this study, we examined the ability of histone deacetylase (HDAC) inhibitors to facilitate extinction and attenuate reinstatement of cocaine-induced conditioned place preference (CPP). METHODS: C57BL/6 mice were subject to cocaine-induced CPP using 20 mg/kg dose. To facilitate extinction, mice were administered an HDAC inhibitor following nonreinforced exposure to the conditioned context. To measure persistence, mice were subject to a reinstatement test using 10 mg/kg dose of cocaine. RESULTS: We demonstrate that HDAC inhibition during extinction consolidation can facilitate extinction of cocaine-induced CPP. Animals treated with an HDAC inhibitor extinguished cocaine-induced CPP both more quickly and to a greater extent than did vehicle-treated animals. We also show that the extinction of cocaine seeking via HDAC inhibition modulates extinction learning such that reinstatement behavior is significantly attenuated. Acetylation of histone H3 in the nucleus accumbens following extinction was increased by HDAC inhibition. CONCLUSIONS: This study provides the first evidence that modulation of chromatin modification can facilitate extinction and prevent reinstatement of drug-induced behavioral changes. These findings provide a potential novel approach to the development of treatments that facilitate extinction of drug-seeking behavior.

Epigenetic mechanisms underlying extinction of memory and drug-seeking behavior.

An increasing body of evidence shows that structural modifications of chromatin, the DNA-protein complex that packages genomic DNA, do not only participate in maintaining cellular memory (e.g., cell fate), but they may also underlie the strengthening and maintenance of synaptic connections required for long-term changes in behavior. Accordingly, epigenetics has become a central topic in several neurobiology fields such as memory, drug addiction, and several psychiatric and mental disorders. This interest is justified as dynamic chromatin modifications may provide not only transient but also stable (or even potentially permanent) epigenetic marks to facilitate, maintain, or block transcriptional processes, which in turn may participate in the molecular neural adaptations underlying behavioral changes. Through epigenetic mechanisms the genome may be indexed in response to environmental signals, resulting in specific neural modifications that largely determine the future behavior of an organism. In this review we discuss recent advances in our understanding of how epigenetic mechanisms contribute to the formation of long-term memory and drug-seeking behavior and potentially how to apply that knowledge to the extinction of memory and drug-seeking behavior.