Studies of olfactory learning in Drosophila have provided key insights into the brain mechanisms underlying learning and memory. One type of olfactory learning, olfactory classical conditioning, consists of learning the contingency between an odor with an aversive or appetitive stimulus. This conditioning requires the activity of molecules that can integrate the two types of sensory information, the odorant as the conditioned stimulus and the aversive or appetitive stimulus as the unconditioned stimulus, in brain regions where the neural pathways for the two stimuli intersect. Compelling data indicate that a particular form of adenylyl cyclase functions as a molecular integrator of the sensory information in the mushroom body neurons. The neuronal pathway carrying the olfactory information from the antennal lobes to the mushroom body is well described. Accumulating data now show that some dopaminergic neurons provide information about aversive stimuli and octopaminergic neurons about appetitive stimuli to the mushroom body neurons. Inhibitory inputs from the GABAergic system appear to gate olfactory information to the mushroom bodies and thus control the ability to learn about odors. Emerging data obtained by functional imaging procedures indicate that distinct memory traces form in different brain regions and correlate with different phases of memory. The results from these and other experiments also indicate that cross talk between mushroom bodies and several other brain regions is critical for memory formation.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Prior studies have shown that they regulate numerous physiological processes critical for normal development, cellular growth control, and organismal behavior. Here, we systematically surveyed 134 different miRNAs for roles in olfactory learning and memory formation using "sponge" technology to titrate their activity broadly in the Drosophila melanogaster central nervous system. We identified at least five different miRNAs involved in memory formation or retention from this large screen, including miR-9c, miR-31a, miR-305, miR-974, and miR-980. Surprisingly, the titration of some miRNAs increased memory, while the titration of others decreased memory. We performed more detailed experiments on two miRNAs, miR-974 and miR-31a, by mapping their roles to subpopulations of brain neurons and testing the functional involvement in memory of potential mRNA targets through bioinformatics and a RNA interference knockdown approach. This screen offers an important first step toward the comprehensive identification of all miRNAs and their potential targets that serve in gene regulatory networks important for normal learning and memory.KEYWORDS genetic screen; learning; memory; Drosophila; miRNA I NVOLVED in post-transcriptional gene regulation, microRNAs (miRNAs) are a class of small noncoding RNAs (Bushati and Cohen 2007). Prior studies have shown that they serve numerous biological processes, ranging from development to tumorigenesis (Esquela-Kerscher and Slack 2006;Kloosterman and Plasterk 2006;Krützfeldt and Stoffel 2006;Chang and Mendell 2007). miRNAs are transcribed as primary miRNAs (pri-miRNAs) from isolated genes or the introns of protein-coding genes ("mirtrons") (Filipowicz et al. 2008). miRNAs are under regulatory influences similar to protein-coding genes (Krol et al. 2010). The pri-miRNAs are then cleaved into precursor miRNAs (pre-miRNAs) by the microprocessor Drosha/Pasha protein complex and transported into the cytoplasm by Exportin5 where they mature through the Dicer/Loquacious protein complex into 21-to 24-nucleotide miRNA hairpins. These hairpins are subsequently assembled with Argonaute-containing protein complexes that bind to specific sequences on target messenger RNAs (mRNAs) using primarily a 2-to 8-nucleotide seed region (Bartel 2009). The small size of the target site allows many mRNAs to be recognized and coregulated by each individual miRNA (Bartel 2009). The miRNA complex, once bound, induces post-transcriptional silencing by translational repression and/or mRNA degradation (Filipowicz et al. 2008;Bazzini et al. 2012;Djuranovic et al. 2012).One important biological process that is understudied relative to miRNA function is learning and memory formation. Among the several known epigenetic processes that allow the nervous system to adapt to environmental signals, the miRNA system is thought to provide relatively rapid and analog control in both time and space over the expression of genomic co...

SUMMARY MicroRNAs have been associated with many different biological functions but little is known about their roles in conditioned behavior. We demonstrate that Drosophila miR-980 is a memory suppressor gene functioning in multiple regions of the adult brain. Memory acquisition and stability were both increased by miR-980 inhibition. Whole cell recordings and functional imaging experiments indicated that miR-980 regulates neuronal excitability. We identified the autism susceptibility gene, A2bp1, as an mRNA target for miR-980. A2bp1 levels varied inversely with miR-980 expression; memory performance was directly related to A2bp1 levels. In addition, A2bp1 knockdown reversed the memory gains produced by miR-980 inhibition, consistent with A2bp1 being a downstream target of miR-980 responsible for the memory phenotypes. Our results indicate that miR-980 represses A2bp1 expression to tune the excitable state of neurons, and the overall state of excitability translates to memory impairment or improvement.

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