Although circular RNAs (circRNAs) are enriched in the brain, their relevance for brain function and psychiatric disorders is poorly understood. Here, we show that circHomer1 is inversely associated with relative HOM-ER1B mRNA isoform levels in both the orbitofrontal cortex (OFC) and stem-cell-derived neuronal cultures of subjects with psychiatric disorders. We further demonstrate that in vivo circHomer1 knockdown (KD) within the OFC can inhibit the synaptic expression of Homer1b mRNA. Furthermore, we show that circHomer1 directly binds to Homer1b mRNA and that Homer1b-specific KD increases synaptic circHomer1 levels and improves OFC-mediated behavioral flexibility. Importantly, double circHomer1 and Homer1b in vivo co-KD results in a complete rescue in circHomer1-associated alterations in both chance reversal learning and synaptic gene expression. Lastly, we uncover an RNA-binding protein that can directly bind to circHomer1 and promote its biogenesis. Taken together, our data provide mechanistic insights into the importance of circRNAs in brain function and disease.

Background: Alcohol consumption during pregnancy can produce behavioral and cognitive deficits that persist into adulthood. These include impairments in executive functions, learning, planning, and cognitive flexibility. We have previously shown that moderate prenatal alcohol exposure (PAE) significantly impairs reversal learning, a measure of flexibility mediated across species by different brain areas that include the orbital frontal cortex (OFC). Reversal learning is likewise impaired by genetic or pharmacological inactivation of GluN2B subunit-containing N-methyl-D-aspartate receptors (NMDARs). In the current study, we tested the hypothesis that moderate PAE persistently alters the number and function of GluN2B subunit-containing NMDARs in OFC pyramidal neurons of adult mice. Methods:We used a rodent model of fetal alcohol spectrum disorders and left offspring undisturbed until adulthood. Using whole-cell, patch-clamp recordings, we assessed NMDAR function in slices from 90-to 100-day-old male and female PAE and control mice. Pharmacologically isolated NMDA receptor-mediated evoked excitatory postsynaptic currents (NMDA-eEPSCs) were recorded in the absence and presence of the GluN2B antagonist, Ro25-6981(1 µM). In a subset of littermates, we evaluated the level of GluN2B protein expression in the synaptic fraction using Western blotting technique. Results:Our results indicate that PAE females show significantly larger (~23%) NMDA-eEPSC amplitudes than controls, while PAE induced a significant decrease (~17%) in NMDA-eEPSC current density of pyramidal neurons recorded in slices from male mice. NMDA-eEPSC decay time was not affected in PAE-exposed mice from either sex. The contribution of GluN2B subunit-containing NMDARs to the eEPSCs was not significantly altered by PAE. Moreover, there were no significant changes in protein expression in the synaptic fraction of either PAE males or females.Conclusions: These findings suggest that low-to-moderate PAE modulates NMDAR function in pyramidal neurons in a sex-specific manner, although we did not find evidence that the effect is mediated by dysfunction of synaptic GluN2B subunitcontaining NMDARs.

IntroductionFetal Alcohol Spectrum Disorders (FASD) are the leading cause of preventable developmental disability and are commonly characterized by alterations in executive function. Reversal learning tasks are reliable, cross-species methods for testing a frequently impaired aspect of executive control, behavioral flexibility. Pre-clinical studies commonly require the use of reinforcers to motivate animals to learn and perform the task. While there are several reinforcers available, the most commonly employed are solid (food pellets) and liquid (sweetened milk) rewards. Previous studies have examined the effects of different solid rewards or liquid dietary content on learning in instrumental responding and found that rodents on liquid reward with higher caloric content performed better with increased response and task acquisition rate. The influence of reinforcer type on reversal learning and how this interacts with developmental insults such as prenatal alcohol exposure (PAE) has not been explored.MethodsWe tested whether reinforcer type during learning or reversal would impact an established deficit in PAE mice.ResultsWe found that all male and female mice on liquid reward, regardless of prenatal exposure were better motivated to learn task behaviors during pre-training. Consistent with previous findings, both male and female PAE mice and Saccharine control mice were able to learn the initial stimulus reward associations irrespective of the reinforcer type. During the initial reversal phase, male PAE mice that received pellet rewards exhibited maladaptive perseverative responding whereas male mice that received liquid rewards performed comparable to their control counterparts. Female PAE mice that received either reinforcer types did not exhibit any deficits on behavioral flexibility. Female saccharine control mice that received liquid, but not pellet, rewards showed increased perseverative responding during the early reversal phase.DiscussionThese data suggest that reinforcer type can have a major impact on motivation, and therefore performance, during reversal learning. Highly motivating rewards may mask behavioral deficits seen with more moderately sought rewards and gestational exposure to the non-caloric sweetener, saccharine, can impact behavior motivated by those reinforcers in a sex-dependent manner.

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