Orbitofrontal intronic circular RNA from Nrxn3 mediates reward learning and motivation for reward.

The orbitofrontal cortex (OFC) is a vital component of brain reward circuitry that is important for reward seeking behavior. However, OFC-mediated molecular mechanisms underlying rewarding behavior are understudied. Here, we report the first circular RNA (circRNA) profile associated with appetitive reward and identify regulation of 92 OFC circRNAs by sucrose self-administration. Among these changes, we observed downregulation of circNrxn3, a circRNA originating from neurexin 3 (Nrxn3), a gene involved in synaptogenesis, learning, and memory. Transcriptomic profiling via RNA sequencing and qPCR of the OFC following in vivo knock-down of circNrxn3 revealed differential regulation of genes associated with pathways important for learning and memory and altered splicing of Nrxn3. Furthermore, circNrxn3 knock-down enhanced sucrose self-administration and motivation for sucrose. Using RNA-immunoprecipitation, we report binding of circNrxn3 to the known Nrxn3 splicing factor SAM68. circNrxn3 is the first reported circRNA capable of regulating reward behavior and circNrxn3-mediated interactions with SAM68 may impact subsequent downstream processing of RNAs such as the regulation of gene expression and splicing.

Orbitofrontal cortex microRNAs support long-lasting heroin seeking behavior in male rats.

Recovery from opioid use disorder (OUD) and maintenance of abstinence from opioid use is hampered by perseverant drug cravings that may persist for months after cessation of drug use. Drug cravings can intensify during the abstinence period, a phenomenon referred to as the 'incubation of craving' that has been well-described in preclinical studies. We previously reported that animals that self-administered heroin at a dosage of 0.075 mg/kg/infusion (HH) paired with discrete drug cues displayed robust incubation of heroin craving behavior after 21 days (D) of forced abstinence, an effect that was not observed with a lower dosage (0.03 mg/kg/infusion; HL). Here, we sought to elucidate molecular mechanisms underlying long-term heroin seeking behavior by profiling microRNA (miRNA) pathways in the orbitofrontal cortex (OFC), a brain region that modulates incubation of heroin seeking. miRNAs are small noncoding RNAs with long half-lives that have emerged as critical regulators of drug seeking behavior but their expression in the OFC has not been examined in any drug exposure paradigm. We employed next generation sequencing to detect OFC miRNAs differentially expressed after 21D of forced abstinence between HH and HL animals, and proteomics analysis to elucidate miRNA-dependent translational neuroadaptations. We identified 55 OFC miRNAs associated with incubation of heroin craving, including miR-485-5p, which was significantly downregulated following 21D forced abstinence in HH but not HL animals. We bidirectionally manipulated miR-485-5p in the OFC to demonstrate that miR-485-5p can regulate long-lasting heroin seeking behavior after extended forced abstinence. Proteomics analysis identified 45 proteins selectively regulated in the OFC of HH but not HL animals that underwent 21D forced abstinence, of which 7 were putative miR-485-5p target genes. Thus, the miR-485-5p pathway is dysregulated in animals with a phenotype of persistent heroin craving behavior and OFC miR-485-5p pathways may function to support long-lasting heroin seeking.

Circular RNA regulation and function in drug seeking phenotypes.

Drug overdoses have increased dramatically in the United States over the last decade where they are now the leading cause of accidental death. To develop efficient therapeutic options for decreasing drug consumption and overdose risk, it is critical to understand the neurobiological changes induced by drug exposure. Chronic systemic exposure to all drug classes, including opioids, psychostimulants, nicotine, cannabis, and alcohol, induces profound molecular neuroadaptations within the central nervous system that may reveal crucial information about the lasting effects that these substances impart on brain cells. Transcriptome analyses of messenger RNAs (mRNAs) have identified gene patterns in the brain that result from exposure to various classes of drugs. However, mRNAs represent only a small fraction of the RNA within the cell, and drug exposure also impacts other classes of RNA that are largely understudied, especially circular RNAs. Circular RNAs (circRNAs) are a naturally occurring RNA species formed from back-splicing events during mRNA processing and are enriched in the nervous system. circRNAs are a pleiotropic class of RNAs and have a diverse impact on cellular function, with putative functions including regulation of mRNA transcription, protein translation, microRNA sponging, and sequestration of RNA-binding proteins. Recent studies have demonstrated that circRNAs can modulate cognition and are regulated in the brain in response to drug exposure, yet very few studies have explored the contribution of circRNAs to drug seeking phenotypes. In this review, we will provide an overview of the mechanisms of circRNA function in the cell to highlight how drug-induced circRNA dysregulation may impact the molecular substrates that mediate drug seeking behavior and the current studies that have reported drug-induced dysregulation of circRNAs in the brain. Furthermore, we will discuss how principles of circRNA biology can be adapted to study circRNAs in models of drug exposure and seek to provide further insight into the neurobiology of addiction.

microRNA expression levels in the nucleus accumbens correlate with morphine-taking but not morphine-seeking behaviour in male rats.

A powerful motivation to seek opioids remains after drug cessation and intensifies during extended periods of abstinence. Unfortunately, biomarkers associated with continued drug seeking have not been described. Moreover, previous studies have focused on the effects of early abstinence with little exploration into the long-term drug-induced mechanisms that occur after extended abstinence. Here we demonstrated that 30 days (D) of forced abstinence results in a time-dependent increase in morphine seeking in a rat model of morphine self-administration (SA). We measured expression of known drug-responsive microRNAs (miRNAs) in the nucleus accumbens, an area critical for reward-related plasticity, during early or late abstinence in animals that underwent either a cue-induced relapse test or no relapse test. miRNAs are small noncoding RNAs that represent suitable biomarker candidates due to their long-lasting nature. mir-32-5p levels during early abstinence negatively correlated with active lever pressing in both cue-exposed and cue-naïve animals. mir-1298-5p positively correlated with drug SA history after a relapse test during late abstinence. When animals underwent acute abstinence with no relapse test, mir-1298-5p correlated with drug infusions and active lever pressing during SA. In late abstinence with no relapse test, mir-137-3p negatively correlated with drug infusions. Regulation of mir-32-5p target genes and significant correlation of target gene mRNA with mir-32-5p was observed after abstinence. These results indicate that lasting regulation of miRNA expression is associated with drug intake following morphine SA. In addition, we conclude that the miRNA profile undergoes regulation from early to late abstinence and miRNA expression may indicate past drug history.

Heroin Regulates Orbitofrontal Circular RNAs.

The number of drug overdose deaths involving opioids continues to rise in the United States. Many patients with opioid use disorder (OUD) that seek treatment still experience relapse. Perseverant opioid seeking behaviors represent a major challenge to treating OUD and additional therapeutic development will require insight into opioid-induced neurobiological adaptations. In this study, we explored the regulation of a novel class of RNAs, circular RNAs (circRNAs), by the addictive opioid heroin in the rat orbitofrontal cortex (OFC), a brain region that mediates behavioral responses to rewarding stimuli. Microarray analysis identified 76 OFC circRNAs significantly regulated in male rats after heroin self-administration. We evaluated the specificity of these findings by measuring heroin-associated circRNA expression in female rats after heroin self-administration and in rats that self-administered sucrose. We identify circGrin2b, circUbe2cp, circAnks1a, circAdcy5 and circSlc24A2 as heroin-responsive circRNAs in the OFC. Linear mRNA levels of heroin-associated circRNAs were unchanged except for Grin2b and Adcy5. An integrated bioinformatics analysis of regulated circRNAs identified microRNAs predicted to bind heroin-associated circRNAs and downstream targets of circRNA: microRNA sponging. Thus, heroin regulates the expression of OFC RNA splice variants that circularize and may impact cellular processes that contribute to the neurobiological adaptations that arise from chronic heroin exposure.

Drug-associated cues and drug dosage contribute to increased opioid seeking after abstinence.

Patients with opioid use disorder experience high rates of relapse during recovery, despite successful completion of rehabilitation programs. A key factor contributing to this problem is the long-lasting nature of drug-seeking behavior associated with opioid use. We modeled this behavior in a rat drug self-administration paradigm in which drug-seeking is higher after extended abstinence than during the acute abstinence phase. The goal of this study was to determine the contribution of discrete or discriminative drug cues and drug dosage to time-dependent increases in drug-seeking. We examined heroin-seeking after 2 or 21 days of abstinence from two different self-administration cue-context environments using high or low doses of heroin and matched animals for their drug intake history. When lower dosages of heroin are used in discriminative or discrete cue protocols, drug intake history contributed to drug-seeking after abstinence, regardless of abstinence length. Incubation of opioid craving at higher dosages paired with discrete drug cues was not dependent on drug intake. Thus, interactions between drug cues and drug dosage uniquely determined conditions permissible for incubation of heroin craving. Understanding factors that contribute to long-lasting opioid-seeking can provide essential insight into environmental stimuli and drug-taking patterns that promote relapse after periods of successful abstinence.

Correction: MicroRNA regulation of persistent stress-enhanced memory.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

MicroRNA regulation of persistent stress-enhanced memory.

Disruption of persistent, stress-associated memories is relevant for treating posttraumatic stress disorder (PTSD) and related syndromes, which develop in a subset of individuals following a traumatic event. We previously developed a stress-enhanced fear learning (SEFL) paradigm in inbred mice that produces PTSD-like characteristics in a subset of mice, including persistently enhanced memory and heightened cFos in the basolateral amygdala complex (BLC) with retrieval of the remote (30-day-old) stress memory. Here, the contribution of BLC microRNAs (miRNAs) to stress-enhanced memory was investigated because of the molecular complexity they achieve through their ability to regulate multiple targets simultaneously. We performed small-RNA sequencing (smRNA-Seq) and quantitative proteomics on BLC tissue collected from mice 1 month after SEFL and identified persistently changed microRNAs, including mir-135b-5p, and proteins associated with PTSD-like heightened fear expression. Viral-mediated overexpression of mir-135b-5p in the BLC of stress-resilient animals enhanced remote fear memory expression and promoted spontaneous renewal 14 days after extinction. Conversely, inhibition of BLC mir-135b-5p in stress-susceptible animals had the opposite effect, promoting a resilient-like phenotype. mir-135b-5p is highly conserved across mammals and was detected in post mortem human amygdala, as well as human serum samples. The mir-135b passenger strand, mir-135b-3p, was significantly elevated in serum from PTSD military veterans, relative to combat-exposed control subjects. Thus, miR-135b-5p may be an important therapeutic target for dampening persistent, stress-enhanced memory and its passenger strand a potential biomarker for responsivity to a mir-135-based therapeutic.

microRNA mir-598-3p mediates susceptibility to stress enhancement of remote fear memory.

microRNAs (miRNAs) have emerged as potent regulators of learning, recent memory, and extinction. However, our understanding of miRNAs directly involved in regulating complex psychiatric conditions perpetuated by aberrant memory, such as in posttraumatic stress disorder (PTSD), remains limited. To begin to address the role of miRNAs in persistent memories, we performed small-RNA sequencing on basolateral amygdala (BLA) tissue and identified miRNAs altered by auditory fear conditioning (FC) one month after training. mir-598-3p, a highly conserved miRNA previously unstudied in the brain, was down-regulated in the BLA. Further decreasing BLA mir-598-3p levels did not increase strength of the remote fear memory. Given that stress is a critical component in PTSD, we next assessed the impact of stress and stress-enhanced fear learning (SEFL) on mir-598-3p levels, finding the miRNA is elevated in the BLA of male, but not female, mice susceptible to the effects of stress in SEFL. Accordingly, intra-BLA inhibition of mir-598-3p interfered with expression and extinction of the remote fear memory in male, but not female, mice. This effect could not be attributed to an anxiolytic effect of miRNA inhibition. Finally, bioinformatic analysis following quantitative proteomics on BLA tissue collected 30 d post-SEFL training identified putative mir-598-3p targets and related pathways mediating the differential susceptibility, with evidence for regulation of the actin cytoskeleton, the core mediator of structural plasticity. Taken together, the results suggest BLA mir-598-3p may be recruited by stress to mediate a critical switch from a salient remote fear memory to one that is enhanced and extinction-resistant.

Bioinformatic analysis of long-lasting transcriptional and translational changes in the basolateral amygdala following acute stress.

Stress profoundly impacts the brain and increases the risk of developing a psychiatric disorder. The brain's response to stress is mediated by a number of pathways that affect gene expression and protein function throughout the cell. Understanding how stress achieves such dramatic effects on the brain requires an understanding of the brain's stress response pathways. The majority of studies focused on molecular changes have employed repeated or chronic stress paradigms to assess the long-term consequences of stress and have not taken an integrative genomic and/or proteomic approach. Here, we determined the lasting impact of a single stressful event (restraint) on the broad molecular profile of the basolateral amygdala complex (BLC), a key brain region mediating emotion, memory and stress. Molecular profiling performed thirty days post-restraint consisted of small RNA sequencing, RNA sequencing and quantitative mass spectrometry and identified long-lasting changes in microRNA (miRNA), messenger RNA (mRNA) and proteins. Alignment of the three datasets further delineated the regulation of stress-specific pathways which were validated by qPCR and Western Blot analysis. From this analysis, mir-29a-5p was identified as a putative regulator of stress-induced adaptations in the BLC. Further, a number of predicted mir-29a-5p targets are regulated at the mRNA and protein level. The concerted and long-lasting disruption of multiple molecular pathways in the amygdala by a single stress event is expected to be sufficient to alter behavioral responses to a wide array of future experiences, including exposure to additional stressors.

Susceptibility and Resilience to Posttraumatic Stress Disorder-like Behaviors in Inbred Mice.

BACKGROUND: The limited neurobiological understanding of posttraumatic stress disorder (PTSD) has been partially attributed to the need for improved animal models. Stress-enhanced fear learning (SEFL) in rodents recapitulates many PTSD-associated behaviors, including stress-susceptible and stress-resilient subgroups in outbred rats. Identification of subgroups requires additional behavioral phenotyping, a confound to mechanistic studies. METHODS: We employed a SEFL paradigm in inbred male and female C57BL/6 mice that combines acute stress with fear conditioning to precipitate traumatic-like memories. Extinction and long-term retention of extinction were examined after SEFL. Further characterization of SEFL effects on male mice was performed with additional behavioral tests, determination of regional activation by Fos immunofluorescence, and RNA sequencing of the basolateral amygdala. RESULTS: Stressed animals displayed persistently elevated freezing during extinction. While more uniform in females, SEFL produced male subgroups with differential susceptibility that were identified without posttraining phenotyping. Additional phenotyping of male mice revealed PTSD-associated behaviors, including extinction-resistant fear memory, hyperarousal, generalization, and dysregulated corticosterone in stress-susceptible male mice. Altered Fos activation was also seen in the infralimbic cortex and basolateral amygdala of stress-susceptible male mice after remote memory retrieval. Key behavioral outcomes, including susceptibility, were replicated by two independent laboratories. RNA sequencing of the basolateral amygdala revealed transcriptional divergence between the male subgroups, including genes with reported polymorphic association to patients with PTSD. CONCLUSIONS: This SEFL model provides a tool for development of PTSD therapeutics that is compatible with the growing number of mouse-specific resources. Furthermore, use of an inbred strain allows for investigation into epigenetic mechanisms that are expected to critically regulate susceptibility and resilience.

The potential of epigenetics in stress-enhanced fear learning models of PTSD.

Prolonged distress and dysregulated memory processes are the core features of post-traumatic stress disorder (PTSD) and represent the debilitating, persistent nature of the illness. However, the neurobiological mechanisms underlying the expression of these symptoms are challenging to study in human patients. Stress-enhanced fear learning (SEFL) paradigms, which encompass both stress and memory components in rodents, are emerging as valuable preclinical models of PTSD. Rodent models designed to study the long-term mechanisms of either stress or fear memory alone have identified a critical role for numerous epigenetic modifications to DNA and histone proteins. However, the epigenetic modifications underlying SEFL remain largely unknown. This review will provide a brief overview of the epigenetic modifications implicated in stress and fear memory independently, followed by a description of existing SEFL models and the few epigenetic mechanisms found to date to underlie SEFL. The results of the animal studies discussed here highlight neuroepigenetics as an essential area for future research in the context of PTSD through SEFL studies, because of its potential to identify novel candidates for neurotherapeutics targeting stress-induced pathogenic memories.

Pharmacological Selectivity Within Class I Histone Deacetylases Predicts Effects on Synaptic Function and Memory Rescue.

Histone deacetylases (HDACs) are promising therapeutic targets for neurological and psychiatric disorders that impact cognitive ability, but the relationship between various HDAC isoforms and cognitive improvement is poorly understood, particularly in mouse models of memory impairment. A goal shared by many is to develop HDAC inhibitors with increased isoform selectivity in order to reduce unwanted side effects, while retaining procognitive effects. However, studies addressing this tack at the molecular, cellular and behavioral level are limited. Therefore, we interrogated the biological effects of class I HDAC inhibitors with varying selectivity and assessed a subset of these compounds for their ability to regulate transcriptional activity, synaptic function and memory. The HDAC-1, -2, and -3 inhibitors, RGFP963 and RGFP968, were most effective at stimulating synaptogenesis, while the selective HDAC3 inhibitor, RGFP966, with known memory enhancing abilities, had minimal impact. Furthermore, RGFP963 increased hippocampal spine density, while HDAC3 inhibition was ineffective. Genome-wide gene expression analysis by RNA sequencing indicated that RGFP963 and RGFP966 induce largely distinct transcriptional profiles in the dorsal hippocampus of mature mice. The results of bioinformatic analyses were consistent with RGFP963 inducing a transcriptional program that enhances synaptic efficacy. Finally, RGFP963, but not RGFP966, rescued memory in a mouse model of Alzheimer's Disease. Together, these studies suggest that the specific memory promoting properties of class I HDAC inhibitors may depend on isoform selectivity and that certain pathological brain states may be more receptive to HDAC inhibitors that improve network function by enhancing synapse efficacy.

Neuroepigenetic Regulation of Pathogenic Memories.

Our unique collection of memories determines our individuality and shapes our future interactions with the world. Remarkable advances into the neurobiological basis of memory have identified key epigenetic mechanisms that support the stability of memory. Various forms of epigenetic regulation at the levels of DNA methylation, histone modification, and non-coding RNAs (ncRNAs) can modulate transcriptional and translational events required for memory processes. By changing the cellular profile in the brain's emotional, reward, and memory circuits, these epigenetic modifications have also been linked to perseverant, pathogenic memories. In this review, we will delve into the relevance of epigenetic dysregulation to pathogenic memory mechanisms by focusing on two neuropsychiatric disorders perpetuated by aberrant memory associations: substance use disorder (SUD) and post-traumatic stress disorder (PTSD). As our understanding improves, neuroepigenetic mechanisms may someday be harnessed to develop novel therapeutic targets for the treatment of these chronic, relapsing disorders.

G9a influences neuronal subtype specification in striatum.

Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we found that G9a repression by cocaine occurred in both Drd1-expressing (striatonigral) and Drd2-expressing (striatopallidal) medium spiny neurons. Conditional knockout and overexpression of G9a within these distinct cell types, however, revealed divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicated that such developmental deletion of G9a selectively in Drd2 neurons resulted in the unsilencing of transcriptional programs normally specific to striatonigral neurons and in the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral 'switching' phenotype in mice indicates a new role for G9a in contributing to neuronal subtype identity and suggests a critical function for cell type-specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli.

ELK1 transcription factor linked to dysregulated striatal mu opioid receptor signaling network and OPRM1 polymorphism in human heroin abusers.

BACKGROUND: Abuse of heroin and prescription opiate medications has grown to disturbing levels. Opioids mediate their effects through mu opioid receptors (MOR), but minimal information exists regarding MOR-related striatal signaling relevant to the human condition. The striatum is a structure central to reward and habitual behavior and neurobiological changes in this region are thought to underlie the pathophysiology of addiction disorders. METHODS: We examined molecular mechanisms related to MOR in postmortem human brain striatal specimens from a homogenous European Caucasian population of heroin abusers and control subjects and in an animal model of heroin self-administration. Expression of ets-like kinase 1 (ELK1) was examined in relation to polymorphism of the MOR gene OPRM1 and drug history. RESULTS: A characteristic feature of heroin abusers was decreased expression of MOR and extracellular regulated kinase signaling networks, concomitant with dysregulation of the downstream transcription factor ELK1. Striatal ELK1 in heroin abusers associated with the polymorphism rs2075572 in OPRM1 in a genotype dose-dependent manner and correlated with documented history of heroin use, an effect reproduced in an animal model that emphasizes a direct relationship between repeated heroin exposure and ELK1 dysregulation. A central role of ELK1 was evidenced by an unbiased whole transcriptome microarray that revealed ~20% of downregulated genes in human heroin abusers are ELK1 targets. Using chromatin immune precipitation, we confirmed decreased ELK1 promoter occupancy of the target gene Use1. CONCLUSIONS: ELK1 is a potential key transcriptional regulatory factor in striatal disturbances associated with heroin abuse and relevant to genetic mutation of OPRM1.

Mitochondria, oligodendrocytes and inflammation in bipolar disorder: evidence from transcriptome studies points to intriguing parallels with multiple sclerosis.

Gene expression studies of bipolar disorder (BPD) have shown changes in transcriptome profiles in multiple brain regions. Here we summarize the most consistent findings in the scientific literature, and compare them to data from schizophrenia (SZ) and major depressive disorder (MDD). The transcriptome profiles of all three disorders overlap, making the existence of a BPD-specific profile unlikely. Three groups of functionally related genes are consistently expressed at altered levels in BPD, SZ and MDD. Genes involved in energy metabolism and mitochondrial function are downregulated, genes involved in immune response and inflammation are upregulated, and genes expressed in oligodendrocytes are downregulated. Experimental paradigms for multiple sclerosis demonstrate a tight link between energy metabolism, inflammation and demyelination. These studies also show variabilities in the extent of oligodendrocyte stress, which can vary from a downregulation of oligodendrocyte genes, such as observed in psychiatric disorders, to cell death and brain lesions seen in multiple sclerosis. We conclude that experimental models of multiple sclerosis could be of interest for the research of BPD, SZ and MDD.

Vascular endothelial growth factor is upregulated by L-dopa in the parkinsonian brain: implications for the development of dyskinesia.

Angiogenesis and increased permeability of the blood-brain barrier have been reported to occur in animal models of Parkinson's disease and l-dopa-induced dyskinesia, but the significance of these phenomena has remained unclear. Using a validated rat model of l-dopa-induced dyskinesia, this study demonstrates that chronic treatment with l-dopa dose dependently induces the expression of vascular endothelial growth factor in the basal ganglia nuclei. Vascular endothelial growth factor was abundantly expressed in astrocytes and astrocytic processes in the proximity of blood vessels. When co-administered with l-dopa, a small molecule inhibitor of vascular endothelial growth factor signalling significantly attenuated the development of dyskinesia and completely blocked the angiogenic response and associated increase in blood-brain barrier permeability induced by the treatment. The occurrence of angiogenesis and vascular endothelial growth factor upregulation was verified in post-mortem basal ganglia tissue from patients with Parkinson's disease with a history of dyskinesia, who exhibited increased microvascular density, microvascular nestin expression and an upregulation of vascular endothelial growth factor messenger ribonucleic acid. These congruent findings in the rat model and human patients indicate that vascular endothelial growth factor is implicated in the pathophysiology of l-dopa-induced dyskinesia and emphasize an involvement of the microvascular compartment in the adverse effects of l-dopa pharmacotherapy in Parkinson's disease.