Identification of the hybrid gene LILRB5-3 by long-read sequencing and implication of its novel signaling function.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) on human chromosome 19q13.4 encode 11 immunoglobulin superfamily receptors, exhibiting genetic diversity within and between human populations. Among the LILR genes, the genomic region surrounding LILRB3 and LILRA6 has yet to be fully characterized due to their significant sequence homology, which makes it difficult to differentiate between them. To examine the LILRB3 and LILRA6 genomic region, a tool named JoGo-LILR CN Caller, which can call copy number from short-read whole genome sequencing (srWGS) data, was applied to an extensive international srWGS dataset comprising 2,504 samples. During this process, a previously unreported loss of both LILRB3 and LILRA6 was detected in three samples. Using long-read sequencing of these samples, we have discovered a novel large deletion (33,692 bp) in the LILRB3 and LILRA6 genomic regions in the Japanese population. This deletion spanned three genes, LILRB3, LILRA6, and LILRB5, resulting in LILRB3 exons 12-13 being located immediately downstream of LILRB5 exons 1-12 with the loss of LILRA6, suggesting the potential expression of a hybrid gene between LILRB5 and LILRB3 (LILRB5-3). Transcription and subsequent translation of the LILRB5-3 hybrid gene were also verified. The hybrid junction was located within the intracellular domain, resulting in an LILRB5 extracellular domain fused to a partial LILRB3 intracellular domain with three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), suggesting that LILRB5-3 acquired a novel signaling function. Further application of the JoGo-LILR tool to srWGS samples suggested the presence of the LILRB5-3 hybrid gene in the CEU population. Our findings provide insight into the genetic and functional diversity of the LILR family.

Neural control of redox response and microbiota-triggered inflammation in Drosophila gut.

Background: The neural system plays a critical role in controlling gut immunity, and the gut microbiota contributes to this process. However, the roles and mechanisms of gut-brain-microbiota interactions remain unclear. To address this issue, we employed Drosophila as a model organism. We have previously shown that NP3253 neurons, which are connected to the brain and gut, are essential for resistance to oral bacterial infections. Here, we aimed to investigate the role of NP3253 neurons in the regulation of gut immunity. Methods: We performed RNA-seq analysis of the adult Drosophila gut after genetically inactivating the NP3253 neurons. Flies were reared under oral bacterial infection and normal feeding conditions. In addition, we prepared samples under germ-free conditions to evaluate the role of the microbiota in gut gene expression. We knocked down the genes regulated by NP3253 neurons and examined their susceptibility to oral bacterial infections. Results: We found that immune-related gene expression was upregulated in NP3253 neuron-inactivated flies compared to the control. However, this upregulation was abolished in axenic flies, suggesting that the immune response was abnormally activated by the microbiota in NP3253 neuron-inactivated flies. In addition, redox-related gene expression was downregulated in NP3253 neuron-inactivated flies, and this downregulation was also observed in axenic flies. Certain redox-related genes were required for resistance to oral bacterial infections, suggesting that NP3253 neurons regulate the redox responses for gut immunity in a microbiota-independent manner. Conclusion: These results show that NP3253 neurons regulate the appropriate gene expression patterns in the gut and contribute to maintain homeostasis during oral infections.

The fruit fly acetyltransferase chameau promotes starvation resilience at the expense of longevity.

Proteins involved in cellular metabolism and molecular regulation can extend lifespan of various organisms in the laboratory. However, any improvement in aging would only provide an evolutionary benefit if the organisms were able to survive under non-ideal conditions. We have previously shown that Drosophila melanogaster carrying a loss-of-function allele of the acetyltransferase chameau (chm) has an increased healthy lifespan when fed ad libitum. Here, we show that loss of chm and reduction in its activity results in a substantial reduction in weight and a decrease in starvation resistance. This phenotype is caused by failure to properly regulate the genes and proteins required for energy storage and expenditure. The previously observed increase in survival time thus comes with the inability to prepare for and cope with nutrient stress. As the ability to survive in environments with restricted food availability is likely a stronger evolutionary driver than the ability to live a long life, chm is still present in the organism's genome despite its apparent negative effect on lifespan.

A descending inhibitory mechanism of nociception mediated by an evolutionarily conserved neuropeptide system in Drosophila.

Nociception is a neural process that animals have developed to avoid potentially tissue-damaging stimuli. While nociception is triggered in the peripheral nervous system, its modulation by the central nervous system is a critical process in mammals, whose dysfunction has been extensively implicated in chronic pain pathogenesis. The peripheral mechanisms of nociception are largely conserved across the animal kingdom. However, it is unclear whether the brain-mediated modulation is also conserved in non-mammalian species. Here, we show that Drosophila has a descending inhibitory mechanism of nociception from the brain, mediated by the neuropeptide Drosulfakinin (DSK), a homolog of cholecystokinin (CCK) that plays an important role in the descending control of nociception in mammals. We found that mutants lacking dsk or its receptors are hypersensitive to noxious heat. Through a combination of genetic, behavioral, histological, and Ca2+ imaging analyses, we subsequently revealed neurons involved in DSK-mediated nociceptive regulation at a single-cell resolution and identified a DSKergic descending neuronal pathway that inhibits nociception. This study provides the first evidence for a descending modulatory mechanism of nociception from the brain in a non-mammalian species that is mediated by the evolutionarily conserved CCK system, raising the possibility that the descending inhibition is an ancient mechanism to regulate nociception.

Avoiding harm is fundamental for the survival of animals. Nerve cells called nociceptors can detect potential damage, such as extreme temperatures, sharp objects and certain chemicals. In humans, this detection ­ known as nociception ­ leads to signals travelling from nociceptors through the spinal cord to the brain, which perceives them as pain. Mammals such as humans and rodents can inhibit nociception by sending signals from the brain to the spinal cord to dampen pain. This top-down dampening process is believed to play a crucial role in regulating pain in mammals, and it has been implicated in the development of chronic pain. It was not known whether non-mammalian animals shared this inhibitory pathway. However, previous work had shown that fruit fly produce a molecule called Drosulfakinin, which is similar to the chemical that mammals use in the top-down signalling pathway which controls pain. To determine the role of Drosulfakinin in controlling fly nociception, Oikawa et al. manipulated its activity ­ and the activity of related genes ­ in specific neurons in the fruit fly nervous system. Without Drosulfakinin, fly larvae were more sensitive to heat exposure, suggesting that this molecule is required to inhibit nociception. Further experiments showed that Drosulfakinin is present only in the brain of fly larvae and activation of its signaling lowers the activity of neurons that transmit nociceptive signals in the insect equivalent of the spinal cord. This confirms that insect brains can dampen nociception via a top-down pathway, using a similar molecule to mammals. The findings provide an important foundation for pain studies using non-mammalian animals. The ability to manipulate nociception using genetic techniques in flies offers a powerful tool to understand the top-down process of controlling pain. This result also raises the possibility that this shared top-down inhibition mechanism may have developed over 550 million years ago, which could lead to further research into how nociception and pain regulation systems evolved.

Female reproductive dormancy in Drosophila is regulated by DH31-producing neurons projecting into the corpus allatum.

Female insects can enter reproductive diapause, a state of suspended egg development, to conserve energy under adverse environments. In many insects, including the fruit fly, Drosophila melanogaster, reproductive diapause, also frequently called reproductive dormancy, is induced under low-temperature and short-day conditions by the downregulation of juvenile hormone (JH) biosynthesis in the corpus allatum (CA). In this study, we demonstrate that neuropeptide Diuretic hormone 31 (DH31) produced by brain neurons that project into the CA plays an essential role in regulating reproductive dormancy by suppressing JH biosynthesis in adult D. melanogaster. The CA expresses the gene encoding the DH31 receptor, which is required for DH31-triggered elevation of intracellular cAMP in the CA. Knocking down Dh31 in these CA-projecting neurons or DH31 receptor in the CA suppresses the decrease of JH titer, normally observed under dormancy-inducing conditions, leading to abnormal yolk accumulation in the ovaries. Our findings provide the first molecular genetic evidence demonstrating that CA-projecting peptidergic neurons play an essential role in regulating reproductive dormancy by suppressing JH biosynthesis.

On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide.

Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. We used the jellyfish Cladonema to address this question. Our combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, we found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. Our results suggest that the satiety signaling systems of diverse animals share an ancient origin.

Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dß1, and Dß2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dß3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dß3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Nutrient responding peptide hormone CCHamide-2 consolidates appetitive memory.

CCHamide-2 (CCHa2) is a protostome excitatory peptide ortholog known for various arthropod species. In fruit flies, CCHa2 plays a crucial role in the endocrine system, allowing peripheral tissue to communicate with the central nervous system to ensure proper development and the maintenance of energy homeostasis. Since the formation of odor-sugar associative long-term memory (LTM) depends on the nutrient status in an animal, CCHa2 may play an essential role in linking memory and metabolic systems. Here we show that CCHa2 signals are important for consolidating appetitive memory by acting on the rewarding dopamine neurons. Genetic disruption of CCHa2 using mutant strains abolished appetitive LTM but not short-term memory (STM). A post-learning thermal suppression of CCHa2 expressing cells impaired LTM. In contrast, a post-learning thermal activation of CCHa2 cells stabilized STM induced by non-nutritious sugar into LTM. The receptor of CCHa2, CCHa2-R, was expressed in a subset of dopamine neurons that mediate reward for LTM. In accordance, the receptor expression in these dopamine neurons was required for LTM specifically. We thus concluded that CCHa2 conveys a sugar nutrient signal to the dopamine neurons for memory consolidation. Our finding establishes a direct interplay between brain reward and the putative endocrine system for long-term energy homeostasis.

The sugar-responsive enteroendocrine neuropeptide F regulates lipid metabolism through glucagon-like and insulin-like hormones in Drosophila melanogaster.

The enteroendocrine cell (EEC)-derived incretins play a pivotal role in regulating the secretion of glucagon and insulins in mammals. Although glucagon-like and insulin-like hormones have been found across animal phyla, incretin-like EEC-derived hormones have not yet been characterised in invertebrates. Here, we show that the midgut-derived hormone, neuropeptide F (NPF), acts as the sugar-responsive, incretin-like hormone in the fruit fly, Drosophila melanogaster. Secreted NPF is received by NPF receptor in the corpora cardiaca and in insulin-producing cells. NPF-NPFR signalling resulted in the suppression of the glucagon-like hormone production and the enhancement of the insulin-like peptide secretion, eventually promoting lipid anabolism. Similar to the loss of incretin function in mammals, loss of midgut NPF led to significant metabolic dysfunction, accompanied by lipodystrophy, hyperphagia, and hypoglycaemia. These results suggest that enteroendocrine hormones regulate sugar-dependent metabolism through glucagon-like and insulin-like hormones not only in mammals but also in insects.

Presynaptic inhibition of dopamine neurons controls optimistic bias.

Regulation of reward signaling in the brain is critical for appropriate judgement of the environment and self. In Drosophila, the protocerebral anterior medial (PAM) cluster dopamine neurons mediate reward signals. Here, we show that localized inhibitory input to the presynaptic terminals of the PAM neurons titrates olfactory reward memory and controls memory specificity. The inhibitory regulation was mediated by metabotropic gamma-aminobutyric acid (GABA) receptors clustered in presynaptic microdomain of the PAM boutons. Cell type-specific silencing the GABA receptors enhanced memory by augmenting internal reward signals. Strikingly, the disruption of GABA signaling reduced memory specificity to the rewarded odor by changing local odor representations in the presynaptic terminals of the PAM neurons. The inhibitory microcircuit of the dopamine neurons is thus crucial for both reward values and memory specificity. Maladaptive presynaptic regulation causes optimistic cognitive bias.

A population of neurons that produce hugin and express the diuretic hormone 44 receptor gene projects to the corpora allata in Drosophila melanogaster.

The corpora allata (CA) are essential endocrine organs that biosynthesize and secrete the sesquiterpenoid hormone, namely juvenile hormone (JH), to regulate a wide variety of developmental and physiological events in insects. CA are directly innervated with neurons in many insect species, implying the innervations to be important for regulating JH biosynthesis. Although this is also true for the model organism Drosophila melanogaster, neurotransmitters produced in the CA-projecting neurons are yet to be identified. In this study on D. melanogaster, we aimed to demonstrate that a subset of neurons producing the neuropeptide hugin, the invertebrate counterpart of the vertebrate neuromedin U, directly projects to the adult CA. A synaptic vesicle marker in the hugin neurons was observed at their axon termini located on the CA, which were immunolabeled with a newly-generated antibody to the JH biosynthesis enzyme JH acid O-methyltransferase. We also found the CA-projecting hugin neurons to likely express a gene encoding the specific receptor for diuretic hormone 44 (Dh44). Moreover, our data suggest that the CA-projecting hugin neurons have synaptic connections with the upstream neurons producing Dh44. Unexpectedly, the inhibition of CA-projecting hugin neurons did not significantly alter the expression levels of the JH-inducible gene Krüppel-homolog 1, which implies that the CA-projecting neurons are not involved in JH biosynthesis but rather in other known biological processes. This is the first study to identify a specific neurotransmitter of the CA-projecting neurons in D. melanogaster, and to anatomically characterize a neuronal pathway of the CA-projecting neurons and their upstream neurons.

Drosophila acquires seconds-scale rhythmic behavior.

Detection of the temporal structure of stimuli is crucial for prediction. While perception of interval timing is relevant for immediate behavioral adaptations, it has scarcely been investigated, especially in invertebrates. Here, we examined whether the fruit fly, Drosophila melanogaster, can acquire rhythmic behavior in the range of seconds. To this end, we developed a novel temporal conditioning paradigm utilizing repeated electric shocks. Combined automatic behavioral annotation and time-frequency analysis revealed that behavioral rhythms continued after cessation of the shocks. Furthermore, we found that aging impaired interval timing. This study thus not only demonstrates the ability of insects to acquire behavioral rhythms of a few seconds, but highlights a life-course decline of temporal coordination, which is also common in mammals.

Voluntary intake of psychoactive substances is regulated by the dopamine receptor Dop1R1 in Drosophila.

Dysregulated motivation to consume psychoactive substances leads to addictive behaviors that often result in serious health consequences. Understanding the neuronal mechanisms that drive drug consumption is crucial for developing new therapeutic strategies. The fruit fly Drosophila melanogaster offers a unique opportunity to approach this problem with a battery of sophisticated neurogenetic tools available, but how they consume these drugs remains largely unknown. Here, we examined drug self-administration behavior of Drosophila and the underlying neuronal mechanisms. We measured the preference of flies for five different psychoactive substances using a two-choice feeding assay and monitored its long-term changes. We found that flies show acute preference for ethanol and methamphetamine, but not for cocaine, caffeine or morphine. Repeated intake of ethanol, but not methamphetamine, increased over time. Preference for methamphetamine and the long-term escalation of ethanol preference required the dopamine receptor Dop1R1 in the mushroom body. The protein level of Dop1R1 increased after repeated intake of ethanol, but not methamphetamine, which correlates with the acquired preference. Genetic overexpression of Dop1R1 enhanced ethanol preference. These results reveal a striking diversity of response to individual drugs in the fly and the role of dopamine signaling and its plastic changes in controlling voluntary intake of drugs.

Mushroom body output differentiates memory processes and distinct memory-guided behaviors.

The mushroom body (MB) of Drosophila melanogaster has multiple functions in controlling memory and behavior.1-9 However, circuit mechanisms that generate this functional diversity are largely unclear. Here, we systematically probed the behavioral contribution of each type of MB output neuron (MBON) by blocking during acquisition, retention, or retrieval of reward or punishment memories. We evaluated the contribution using two conditioned responses: memory-guided odor choice and odor source attraction. Quantitative analysis revealed that these conditioned odor responses are controlled by different sets of MBONs. We found that the valence of memory, rather than the transition of memory steps, has a larger impact on the patterns of required MBONs. Moreover, we found that the glutamatergic MBONs forming recurrent circuits commonly contribute to appetitive memory acquisition, suggesting a pivotal role of this circuit motif for reward processing. Our results provide principles how the MB output circuit processes associative memories of different valence and controls distinct memory-guided behaviors.

Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster.

Stem cells fuel the development and maintenance of tissues. Many studies have addressed how local signals from neighboring niche cells regulate stem cell identity and their proliferative potential. However, the regulation of stem cells by tissue-extrinsic signals in response to environmental cues remains poorly understood. Here we report that efferent octopaminergic neurons projecting to the ovary are essential for germline stem cell (GSC) increase in response to mating in female Drosophila. The neuronal activity of the octopaminergic neurons is required for mating-induced GSC increase as they relay the mating signal from sex peptide receptor-positive cholinergic neurons. Octopamine and its receptor Oamb are also required for mating-induced GSC increase via intracellular Ca2+ signaling. Moreover, we identified Matrix metalloproteinase-2 as a downstream component of the octopamine-Ca2+ signaling to induce GSC increase. Our study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues through stem cell niche signaling.

Stem cells have the unique ability to mature into the various, specialized groups of cells required for organisms to work properly. Local signals released by the tissues immediately surrounding stem cells usually trigger this specialization process. However, recent studies have revealed that external signals, such as hormones or neurotransmitters (the chemicals used by nerve cells to communicate), can also control the fate of stem cells. This is particularly the case during development, or in response to events such as injury. In the right conditions, germline stem cells can specialize into the egg or sperm required for many animals to reproduce. In fruit flies for example, the semen contains proteins that activate a cascade of molecular events in the female nervous system, ultimately resulting in female germline stem cells multiplying in the ovaries after mating. Yet, exactly how this process takes place was still unclear. To investigate this question, Yoshinari et al. focused on nerve cells in the fruit fly ovary which produce a neurotransmitter called octopamine. The experiments assessed changes in the ovaries of female fruit flies after mating, piecing together the sequence of events that activate germline stem cells. This showed that first, mating triggers the release of octopamine from the nerve cells. In turn, this activates a protein called Oamb, which is studded through the membrane of cells present around germline stem cells. Turning on Oamb prompts a cascade of molecular events which include an enzyme called Matrix metalloproteinase 2 regulating the signal sent from the local environment to germline stem cells. As mammals use a neurotransmitter similar to octopamine, future fruit fly studies could shed light on how neurotransmitters activate stem cells in other animals. Ultimately, unravelling the way external signals trigger the specialization process may offer insight into how diseases arise from uncontrolled stem cell activity.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

The Corazonin-PTTH Neuronal Axis Controls Systemic Body Growth by Regulating Basal Ecdysteroid Biosynthesis in Drosophila melanogaster.

Steroid hormones play key roles in development, growth, and reproduction in various animal phyla [1]. The insect steroid hormone, ecdysteroid, coordinates growth and maturation, represented by molting and metamorphosis [2]. In Drosophila melanogaster, the prothoracicotropic hormone (PTTH)-producing neurons stimulate peak levels of ecdysteroid biosynthesis for maturation [3]. Additionally, recent studies on PTTH signaling indicated that basal levels of ecdysteroid negatively affect systemic growth prior to maturation [4-8]. However, it remains unclear how PTTH signaling is regulated for basal ecdysteroid biosynthesis. Here, we report that Corazonin (Crz)-producing neurons regulate basal ecdysteroid biosynthesis by affecting PTTH neurons. Crz belongs to gonadotropin-releasing hormone (GnRH) superfamily, implying an analogous role in growth and maturation [9]. Inhibition of Crz neuronal activity increased pupal size, whereas it hardly affected pupariation timing. This phenotype resulted from enhanced growth rate and a delay in ecdysteroid elevation during the mid-third instar larval (L3) stage. Interestingly, Crz receptor (CrzR) expression in PTTH neurons was higher during the mid- than the late-L3 stage. Silencing of CrzR in PTTH neurons increased pupal size, phenocopying the inhibition of Crz neuronal activity. When Crz neurons were optogenetically activated, a strong calcium response was observed in PTTH neurons during the mid-L3, but not the late-L3, stage. Furthermore, we found that octopamine neurons contact Crz neurons in the subesophageal zone (SEZ), transmitting signals for systemic growth. Together, our results suggest that the Crz-PTTH neuronal axis modulates ecdysteroid biosynthesis in response to octopamine, uncovering a regulatory neuroendocrine system in the developmental transition from growth to maturation.

Dopamine Receptor Dop1R2 Stabilizes Appetitive Olfactory Memory through the Raf/MAPK Pathway in Drosophila.

In Drosophila, dopamine signaling to the mushroom body intrinsic neurons, Kenyon cells (KCs), is critical to stabilize olfactory memory. Little is known about the downstream intracellular molecular signaling underlying memory stabilization. Here we address this question in the context of sugar-rewarded olfactory long-term memory (LTM). We show that associative training increases the phosphorylation of MAPK in KCs, via Dop1R2 signaling. Consistently, the attenuation of Dop1R2, Raf, or MAPK expression in KCs selectively impairs LTM, but not short-term memory. Moreover, we show that the LTM deficit caused by the knockdown of Dop1R2 can be rescued by expressing active Raf in KCs. Thus, the Dop1R2/Raf/MAPK pathway is a pivotal downstream effector of dopamine signaling for stabilizing appetitive olfactory memory.SIGNIFICANCE STATEMENT Dopaminergic input to the Kenyon cells (KCs) is pivotal to stabilize memory in Drosophila This process is mediated by dopamine receptors like Dop1R2. Nevertheless, little is known for its underlying molecular mechanism. Here we show that the Raf/MAPK pathway is specifically engaged in appetitive long-term memory in KCs. With combined biochemical and behavioral experiments, we reveal that activation of the Raf/MAPK pathway is regulated through Dop1R2, shedding light on how dopamine modulates intracellular signaling for memory stabilization.

Environmental Light Is Required for Maintenance of Long-Term Memory in Drosophila.

Long-term memory (LTM) is stored as functional modifications of relevant neural circuits in the brain. A large body of evidence indicates that the initial establishment of such modifications through the process known as memory consolidation requires learning-dependent transcriptional activation and de novo protein synthesis. However, it remains poorly understood how the consolidated memory is maintained for a long period in the brain, despite constant turnover of molecular substrates. Using the Drosophila courtship conditioning assay of adult males as a memory paradigm, here, we show that in Drosophila, environmental light plays a critical role in LTM maintenance. LTM is impaired when flies are kept in constant darkness (DD) during the memory maintenance phase. Because light activates the brain neurons expressing the neuropeptide pigment-dispersing factor (Pdf), we examined the possible involvement of Pdf neurons in LTM maintenance. Temporal activation of Pdf neurons compensated for the DD-dependent LTM impairment, whereas temporal knockdown of Pdf during the memory maintenance phase impaired LTM in light/dark cycles. Furthermore, we demonstrated that the transcription factor cAMP response element-binding protein (CREB) is required in the memory center, namely, the mushroom bodies (MBs), for LTM maintenance, and Pdf signaling regulates light-dependent transcription via CREB. Our results demonstrate for the first time that universally available environmental light plays a critical role in LTM maintenance by activating the evolutionarily conserved memory modulator CREB in MBs via the Pdf signaling pathway.SIGNIFICANCE STATEMENT Temporary memory can be consolidated into long-term memory (LTM) through de novo protein synthesis and functional modifications of neuronal circuits in the brain. Once established, LTM requires continual maintenance so that it is kept for an extended period against molecular turnover and cellular reorganization that may disrupt memory traces. How is LTM maintained mechanistically? Despite the critical importance of LTM maintenance, its molecular and cellular underpinnings remain elusive. This study using Drosophila is significant because it revealed for the first time in any organism that universally available environmental light plays an essential role in LTM maintenance. Interestingly, light does so by activating the evolutionarily conserved transcription factor cAMP response element-binding protein via peptidergic signaling.