Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems.

Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.

Prolonged Withdrawal From Escalated Oxycodone Is Associated With Increased Expression of Glutamate Receptors in the Rat Hippocampus.

People suffering from opioid use disorder (OUD) exhibit cognitive dysfunctions. Here, we investigated potential changes in the expression of glutamate receptors in rat hippocampi at 2 h and 31 days after the last session of oxycodone self-administration (SA). RNA extracted from the hippocampus was used in quantitative polymerase chain reaction analyses. Rats, given long-access (9 h per day) to oxycodone (LgA), took significantly more drug than rats exposed to short-access (3 h per day) (ShA). In addition, LgA rats could be further divided into higher oxycodone taking (LgA-H) or lower oxycodone taking (LgA-L) groups, based on a cut-off of 50 infusions per day. LgA rats, but not ShA, rats exhibited incubation of oxycodone craving. In addition, LgA rats showed increased mRNA expression of GluA1-3 and GluN2a-c subunits as well as Grm3, Grm5, Grm6, and Grm8 subtypes of glutamate receptors after 31 days but not after 2 h of stopping the SA experiment. Changes in GluA1-3, Grm6, and Grm8 mRNA levels also correlated with increased lever pressing (incubation) after long periods of withdrawal from oxycodone. More studies are needed to elucidate the molecular mechanisms involved in altering the expression of these receptors during withdrawal from oxycodone and/or incubation of drug seeking.

Escalated Oxycodone Self-Administration Causes Differential Striatal mRNA Expression of FGFs and IEGs Following Abstinence-Associated Incubation of Oxycodone Craving.

Addiction to prescribed opioids including oxycodone has reached tragic levels. Herein, we investigated the relevance of fibroblast growth factors (FGFs) and immediate early genes (IEGs) to withdrawal-induced incubation of drug craving following escalated oxycodone self-administration (SA). Rats were trained to self-administer oxycodone for 4 weeks. Seeking tests were performed at various intervals during 1 month of drug withdrawal. Rats were euthanized 1 day after the last test and nucleus accumbens and dorsal striata were dissected for use in PCR analyses. Rats given long access (LgA, 9 h), but not short access (ShA, 3 h) to drug escalated their oxycodone intake and exhibited incubation of oxycodone seeking during withdrawal. These rats exhibited dose-dependent increases in fgf2 expression in the dorsal striatum. Fgfr2 expression was also significantly increased in the striatum in LgA, but not ShA groups. Similarly, striatal c-fos and junB mRNA levels showed greater increases in LgA rats. The observations that fgf mRNA levels were more altered in the dorsal striatum than in the NAc of LgA rats suggest that changes in striatal FGF expression may be more salient to incubation of oxycodone craving than alterations in the NAc. Targeting FGF signaling pathways might offer novel strategies against opioid addiction.