•15 different PDE10A transcripts are now cataloged in the human brain•The novel PDE10A transcripts have unique 5′ sequences but retain identical sequences in the carboxyl-half of the protein•Novel PDE10A exons are conserved in non-human primate species and are rarely found in other mammals•108 “intronic” SNPs, of which 78% are rare variants, were found in the novel PDE10A exons, most residing in the 5’UTRsPDE10A is a cAMP/cGMP phosphodiesterase important in signal transduction within medium spiny neurons of the human striatum. This gene region has been associated with bipolar disorder via case-control and linkage studies. The three most studied human PDE10A isoforms differ in both their N-termini and trafficking within the cell with PDE10A2 found predominantly at the plasma membrane and PDE10A1 and PDE10A19 remaining primarily within the cytosol. RNA-sequencing and 5′ RLM-RACE studies of the human putamen and caudate nucleus revealed 16 new exons and 12 novel transcripts of PDE10A, 3 of which are predicted to produce proteins with unique N-termini. The novel first exons of these transcripts are highly conserved in non-human primate species and are rarely found in other mammals. One hundred and eight previously classified intronic SNPs were found within the novel PDE10A exons of which 78% were classified as rare variants. Since most of the rare variants localize to 5′ UTR regions, they may influence PDE10A transcription, translation, or mRNA stability. Dysregulation of cAMP signaling has been proposed as a cause of bipolar disorder and PDE10A inhibitors have been investigated as potential therapeutics for schizophrenia. Understanding the mechanisms contributing to PDE10A expression in the human striatum may provide evidence linking this gene to the phenotypes observed in neuropsychiatric disorders.

•The dopamine receptor dDA1 couples to Gs for intracellular signaling•The dopamine receptor Damb couples to Gq for intracellular signaling•Gαq knockdown with RNAi in the mushroom bodies inhibits forgetting•Dopamine spurs acquisition through dDA1/Gs and forgetting through Damb/GqPrior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cyclic AMP (cAMP) accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. Here, we surveyed the spectrum of G protein coupling of dDA1 and Damb, and we confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca2+ signaling with greater efficiency and dopamine sensitivity. The knockdown of Gαq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. Our findings identify a Damb/Gq-signaling pathway that stimulates forgetting and resolves the opposing effects of dopamine on acquisition and forgetting.Download high-res image (146KB)Download full-size image

Pioneering research studies, beginning with those using Drosophila, have identified several molecular and cellular mechanisms for active forgetting. The currently known mechanisms for active forgetting include neurogenesis-based forgetting, interference-based forgetting, and intrinsic forgetting, the latter term describing the brain’s chronic signaling systems that function to slowly degrade molecular and cellular memory traces. The best-characterized pathway for intrinsic forgetting includes “forgetting cells” that release dopamine onto engram cells, mobilizing a signaling pathway that terminates in the activation of Rac1/Cofilin to effect changes in the actin cytoskeleton and neuron/synapse structure. Intrinsic forgetting may be the default state of the brain, constantly promoting memory erasure and competing with processes that promote memory stability like consolidation. A better understanding of active forgetting will provide insights into the brain’s memory management system and human brain disorders that alter active forgetting mechanisms.

Bipolar disorder is a highly heritable neuropsychiatric disorder affecting nearly 2.5% of the population. Prior genetic studies identified a panel of common and rare single-nucleotide polymorphisms associated with the disease that map to the first intron of the PDE10A gene. RNA sequencing of striatal brain tissue from bipolar and healthy control subjects identified a novel transcript of PDE10A, named PDE10A19, that codes for a PDE10A isoform with a unique N terminus. Genomic sequences that can encode the novel N terminus were conserved in other primates but not rodents. The RNA transcript was expressed at equal or greater levels in the human striatum compared with the two annotated transcripts, PDE10A1 and PDE10A2. The PDE10A19 transcript was detected in polysomal fractions; western blotting experiments confirmed that the RNA transcript is translated into protein. Immunocytochemistry studies using transfected mouse striatal and cortical neurons demonstrated that the PDE10A19 protein distributes to the cytosol, like PDE10A1, and unlike PDE10A2, which is associated with plasma membranes. Immunoprecipitation and immunocytochemical experiments revealed that the PDE10A19 isoform interacts physically with PDE10A2 and, when expressed at elevated levels, interferes with the plasma membrane localization of PDE10A2. These studies illustrate the complexity of PDE10A gene expression in the human brain and highlight the need to unravel the gene’s complex and complete coding capabilities along with its transcriptional and translational regulation to guide the development of therapeutic agents that target the protein for the treatment of neuropsychiatric illness.

The genetic basis for bipolar disorder (BPD) is complex with the involvement of multiple genes. As it is well established that cyclic adenosine monophosphate (cAMP) signaling regulates behavior, we tested variants in 29 genes that encode components of this signaling pathway for associations with BPD type I (BPD I) and BPD type II (BPD II). A total of 1172 individuals with BPD I, 516 individuals with BPD II and 1728 controls were analyzed. Single SNP (single-nucleotide polymorphism), haplotype and SNP × SNP interactions were examined for association with BPD. Several statistically significant single-SNP associations were observed between BPD I and variants in the PDE10A gene and between BPD II and variants in the DISC1 and GNAS genes. Haplotype analysis supported the conclusion that variation in these genes is associated with BPD. We followed-up PDE10A’s association with BPD I by sequencing a 23-kb region in 30 subjects homozygous for seven minor allele risk SNPs and discovered eight additional rare variants (minor allele frequency <1%). These single-nucleotide variants were genotyped in 999 BPD cases and 801 controls. We obtained a significant association for these variants in the combined sample using multiple methods for rare variant analysis. After using newly developed methods to account for potential bias from sequencing BPD cases only, the results remained significant. In addition, SNP × SNP interaction studies suggested that variants in several cAMP signaling pathway genes interact to increase the risk of BPD. This report is among the first to use multiple rare variant analysis methods following common tagSNPs associations with BPD.

How the functional activity of the brain is altered during aging to cause age-related memory impairments is unknown. We used functional cellular imaging to monitor two different calcium-based memory traces that underlie olfactory classical conditioning in young and aged Drosophila. Functional imaging of neural activity in the processes of the dorsal paired medial (DPM) and mushroom body neurons revealed that the capacity to form an intermediate-term memory (ITM) trace in the DPM neurons after learning is lost with age, whereas the capacity to form a short-term memory trace in the α′/β′ mushroom body neurons remains unaffected by age. Stimulation of the DPM neurons by activation of a temperature-sensitive cation channel between acquisition and retrieval enhanced ITM in aged but not young flies. These data indicate that the functional state of the DPM neurons is selectively altered with age to cause an age-related impairment of ITM, and demonstrate that altering the excitability of DPM neurons can restore age-related memory impairments.

•RISC is required for activity-dependent and sensory-specific protein translation.•RISC and associated proteins are required for olfactory long-term habituation.•MiR-276a regulates long-term memory by controlling dopamine receptor expression.•Let-7 is necessary for normal development of αβ MBn and learning in adult flies.•MiR-980, a memory suppressor, regulates memory formation through A2bp1.MicroRNAs (miRs) are small non-coding RNAs that regulate protein expression through post-transcriptional mechanisms. They participate in broad aspects of biology from the control of developmental processes to tumorigenesis. Recent studies in Drosophila show that they also regulate activity-dependent and sensory-specific protein expression and support olfactory memory formation. Among the hundreds of miRs described, several have been demonstrated to be required for normal learning, memory, or for the development of neuronal circuits that support memory formation. Fly models of human diseases offer promise of identifying miRs whose expression becomes dysregulated and part of the pathological state, providing models for understanding brain disorders and drug discovery.

•Solute Carrier 22A of Drosophila (DmSLC22A) is a memory suppressor gene•DmSLC22A influences the rate of acquisition of olfactory memories•It is expressed and functions in the dendrites of mushroom body neurons•It functions as a cholinergic transporter to limit neurotransmitter actionsThe mechanisms that constrain memory formation are of special interest because they provide insights into the brain’s memory management systems and potential avenues for correcting cognitive disorders. RNAi knockdown in the Drosophila mushroom body neurons (MBn) of a newly discovered memory suppressor gene, Solute Carrier DmSLC22A, a member of the organic cation transporter family, enhances olfactory memory expression, while overexpression inhibits it. The protein localizes to the dendrites of the MBn, surrounding the presynaptic terminals of cholinergic afferent fibers from projection neurons (Pn). Cell-based expression assays show that this plasma membrane protein transports cholinergic compounds with the highest affinity among several in vitro substrates. Feeding flies choline or inhibiting acetylcholinesterase in Pn enhances memory, an effect blocked by overexpression of the transporter in the MBn. The data argue that DmSLC22A is a memory suppressor protein that limits memory formation by helping to terminate cholinergic neurotransmission at the Pn:MBn synapse.

Functional imaging with genetically encoded calcium and cAMP reporters was used to examine the signal integration underlying learning in Drosophila. Dopamine and octopamine modulated intracellular cAMP in spatially distinct patterns in mushroom body neurons. Pairing of neuronal depolarization with subsequent dopamine application revealed a synergistic increase in cAMP in the mushroom body lobes, which was dependent on the rutabaga adenylyl cyclase. This synergy was restricted to the axons of mushroom body neurons, and occurred only following forward pairing with time intervals similar to those required for behavioral conditioning. In contrast, forward pairing of neuronal depolarization and octopamine produced a subadditive effect on cAMP. Finally, elevating intracellular cAMP facilitated calcium transients in mushroom body neurons, suggesting that cAMP elevation is sufficient to induce presynaptic plasticity. These data suggest that rutabaga functions as a coincidence detector in an intact neuronal circuit, with dopamine and octopamine bidirectionally influencing the generation of cAMP.

•MicroRNA-iab-3p is required during development for normal adult olfactory learning.•MicroRNA-iab-3p is required for the normal development of mushroom body neurons.•A potential functional target for this microRNA is phosphothanolamine synthase.MicroRNAs are small non-coding RNAs that inhibit protein expression post-transcriptionally. They have been implicated in many different physiological processes, but little is known about their individual involvement in learning and memory. We recently identified several miRNAs that either increased or decreased intermediate-term memory when inhibited in the central nervous system, including miR-iab8-3p. We report here a new developmental role for this miRNA. Blocking the expression of miR-iab8-3p during the development of the organism leads to hypertrophy of individual mushroom body neuron soma, a reduction in the field size occupied by axonal projections, and adult intellectual disability. We further identified four potential mRNA targets of miR-iab8-3p whose inhibition modulates intermediate-term memory including ceramide phosphoethanolamine synthase, which may account for the behavioral effects produced by miR-iab8-3p inhibition. Our results offer important new information on a microRNA required for normal neurodevelopment and the capacity to learn and remember normally.Download high-res image (270KB)Download full-size image

Studies using functional cellular imaging of living flies have identified six memory traces that form in the olfactory nervous system after conditioning with odors. These traces occur in distinct nodes of the olfactory nervous system, form and disappear across different windows of time, and are detected in the imaged neurons as increased calcium influx or synaptic release in response to the conditioned odor. Three traces form at or near acquisition and coexist with short-term behavioral memory. One trace forms with a delay after learning and coexists with intermediate-term behavioral memory. Two traces form many hours after acquisition and coexist with long-term behavioral memory. The transient memory traces may support behavior across the time windows of their existence. The experimental approaches for dissecting memory formation in the fly, ranging from the molecular to the systems, make it an ideal system for elucidating the logic by which the nervous system organizes and stores different temporal forms of memory.

Psychological studies in humans and behavioral studies of model organisms suggest that forgetting is a common and biologically regulated process, but the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. Here we show that the bidirectional modulation of a small subset of dopamine neurons (DANs) after olfactory learning regulates the rate of forgetting of both punishing (aversive) and rewarding (appetitive) memories. Two of these DANs, MP1 and MV1, exhibit synchronized ongoing activity in the mushroom body neuropil in alive and awake flies before and after learning, as revealed by functional cellular imaging. Furthermore, while the mushroom-body-expressed dDA1 dopamine receptor is essential for the acquisition of memory, we show that the dopamine receptor DAMB, also highly expressed in mushroom body neurons, is required for forgetting. We propose a dual role for dopamine: memory acquisition through dDA1 signaling and forgetting through DAMB signaling in the mushroom body neurons.Highlights► Dopamine neuron synaptic function regulates forgetting of olfactory memories ► Dopamine neurons exhibit ongoing activity in the mushroom bodies in vivo ► The dopamine receptor DAMB mediates dopamine-based forgetting