Local neurons in the vertebrate retina are instrumental in transforming visual inputs to extract contrast, motion, and color information and in shaping bipolar-to-ganglion cell transmission to the brain. In Drosophila, UV vision is represented by R7 inner photoreceptor neurons that project to the medulla M6 stratum, with relatively little known of this downstream substrate. Here, using R7 terminals as references, we generated a 3D volume model of the M6 stratum, which revealed a retinotopic map for UV representations. Using this volume model as a common 3D framework, we compiled and analyzed the spatial distributions of more than 200 single M6-specific local neurons (M6-LNs). Based on the segregation of putative dendrites and axons, these local neurons were classified into two families, directional and nondirectional. Neurotransmitter immunostaining suggested a signal routing model in which some visual information is relayed by directional M6-LNs from the anterior to the posterior M6 and all visual information is inhibited by a diverse population of nondirectional M6-LNs covering the entire M6 stratum. Our findings suggest that the Drosophila medulla M6 stratum contains diverse LNs that form repeating functional modules similar to those found in the vertebrate inner plexiform layer.

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30-300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.

Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αβ, α'β', and γ neurons. Previously we demonstrated that two parallel pathways mediated ARM consolidation: the serotonergic dorsal paired medial (DPM)-αβ neurons and the octopaminergic anterior paired lateral (APL)-α'β' neurons. This finding prompted us to ask how this composite ARM is retrieved. Here, we showed that blocking the output of αβ neurons and that of α'β' neurons each impaired ARM retrieval, and blocking both simultaneously had an additive effect. Knockdown of radish and octβ2R in αβ and α'β' neurons, respectively, impaired ARM. A combinatorial assay of radish mutant background rsh1 and neurotransmission blockade confirmed that ARM retrieved from α'β' neuron output is independent of radish. We identified MBON-β2β'2a and MBON-β'2mp as the MB output neurons downstream of αβ and α'β' neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, we showed that α'β' neurons could be functionally subdivided into α'β'm neurons required for ARM retrieval, and α'β'ap neurons required for ARM consolidation. Our work demonstrated that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval.

Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

Aggregation of the amyloid-beta-42 (Abeta42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer's disease (AD), and the prevention of Abeta aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Abeta can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Abeta42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Abeta42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Abeta42Arc) and an artificial mutation (Abeta42art) that is known to suppress aggregation and toxicity of Abeta42 in vitro. In the Drosophila brain, Abeta42Arc formed more oligomers and deposits than did wild type Abeta42, while Abeta42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Abeta peptides. Surprisingly, however, Abeta42art caused earlier onset of memory defects than Abeta42. More remarkably, each Abeta induced qualitatively different pathologies. Abeta42Arc caused greater neuron loss than did Abeta42, while Abeta42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Abeta aggregates: Abeta42Arc formed large deposits in the cell body, Abeta42art accumulated preferentially in the neurites, while Abeta42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Abeta42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.

Electrical synapses between neurons, also known as gap junctions, are direct cell membrane channels between adjacent neurons. Gap junctions play a role in the synchronization of neuronal network activity; however, their involvement in cognition has not been well characterized. Three-hour olfactory associative memory in Drosophila has two components: consolidated anesthesia-resistant memory (ARM) and labile anesthesia-sensitive memory (ASM). Here, we show that knockdown of the gap junction gene innexin5 (inx5) in mushroom body (MB) neurons disrupted ARM, while leaving ASM intact. Whole-mount brain immunohistochemistry indicated that INX5 protein was preferentially expressed in the somas, calyxes, and lobes regions of the MB neurons. Adult-stage-specific knockdown of inx5 in αβ neurons disrupted ARM, suggesting a specific requirement of INX5 in αβ neurons for ARM formation. Hyperpolarization of αβ neurons during memory retrieval by expressing an engineered halorhodopsin (eNpHR) also disrupted ARM. Administration of the gap junction blocker carbenoxolone (CBX) reduced the proportion of odor responsive αβ neurons to the training odor 3 hours after training. Finally, the α-branch-specific 3-hour ARM-specific memory trace was also diminished with CBX treatment and in inx5 knockdown flies. Altogether, our results suggest INX5 gap junction channels in αβ neurons for ARM retrieval and also provide a more detailed neuronal mechanism for consolidated memory in Drosophila.

The neurology of male sexuality has been poorly studied owing to difficulties in studying brain circuitry in humans. Dopamine (DA) is essential for both physiological and behavioural responses, including the regulation of sexuality. Previous studies have revealed that alterations in DA synthesis in dopaminergic neurons can induce male-male courtship behaviour, while increasing DA levels in the protocerebral posteriolateral dopaminergic cluster neuron 2ab (PPL2ab) may enhance the intensity of male courtship sustainment in Drosophila. Here we report that changes in the ability of the PPL2ab in the central nervous system (CNS) to produce DA strongly impact male-male courtship in D. melanogaster. Intriguingly, the DA-synthesizing abilities of these neurons appear to affect both the courting activities displayed by male flies and the sex appeal of male flies for other male flies. Moreover, the observed male-male courtship is triggered primarily by target motion, yet chemical cues can replace visual input under dark conditions. This is interesting evidence that courtship responses in male individuals are controlled by PPL2ab neurons in the CNS. Our study provides insight for subsequent studies focusing on sexual circuit modulation by PPL2ab neurons.

Animals rapidly acquire surrounding information to perform the appropriate behavior. Although social learning is more efficient and accessible than self-learning for animals, the detailed regulatory mechanism of social learning remains unknown, mainly because of the complicated information transfer between animals, especially for aversive conditioning information transmission. The current study revealed that, during social learning, the neural circuit in observer flies used to process acquired aversive conditioning information from demonstrator flies differs from the circuit used for self-learned classic aversive conditioning. This aversive information transfer is species dependent. Solitary flies cannot learn this information through social learning, suggesting that this ability is not an innate behavior. Neurons used to process and execute avoidance behavior to escape from electrically shocked flies are all in the same brain region, indicating that the fly brain has a common center for integrating external stimuli with internal states to generate flight behavior.

The possible neurological and biophysical effects of magnetic fields on animals is an area of active study. Here, we report that courtship activity of male Drosophila increases in a magnetic field and that this effect is regulated by the blue light-dependent photoreceptor cryptochrome (CRY). Naïve male flies exhibited significantly increased courtship activities when they were exposed to a ≥ 20-Gauss static magnetic field, compared with their behavior in the natural environment (0 Gauss). CRY-deficient flies, cryb and crym, did not show an increased courtship index in a magnetic field. RNAi-mediated knockdown of cry in cry-GAL4-positive neurons disrupted the increased male courtship activity in a magnetic field. Genetically expressing cry under the control of cry-GAL4 in the CRY-deficient flies restored the increase in male courtship index that occurred in a magnetic field. Interestingly, artificially activating cry-GAL4-expressing neurons, which include large ventral lateral neurons and small ventral lateral neurons, via expression of thermosensitive cation channel dTrpA1, also increased the male courtship index. This enhancement was abolished by the addition of the cry-GAL80 transgene. Our results highlight the phenomenon of increased male courtship activity caused by a magnetic field through CRY-dependent magnetic sensation in CRY expression neurons in Drosophila.

In humans and many other animals, memory consolidation occurs through multiple temporal phases and usually involves more than one neuroanatomical brain system. Genetic dissection of Pavlovian olfactory learning in Drosophila melanogaster has revealed multiple memory phases, but the predominant view holds that all memory phases occur in mushroom body neurons. Here, we demonstrate an acute requirement for NMDA receptors (NMDARs) outside of the mushroom body during long-term memory (LTM) consolidation. Targeted dsRNA-mediated silencing of Nmdar1 and Nmdar2 (also known as dNR1 or dNR2, respectively) in cholinergic R4m-subtype large-field neurons of the ellipsoid body specifically disrupted LTM consolidation, but not retrieval. Similar silencing of functional NMDARs in the mushroom body disrupted an earlier memory phase, leaving LTM intact. Our results clearly establish an anatomical site outside of the mushroom body involved with LTM consolidation, thus revealing both a distributed brain system subserving olfactory memory formation and the existence of a system-level memory consolidation in Drosophila.

Male sexual desire typically declines with ageing. However, our understanding of the neurobiological basis for this phenomenon is limited by our knowledge of the brain circuitry and neuronal pathways controlling male sexual desire. A number of studies across species suggest that dopamine (DA) affects sexual desire. Here we use genetic tools and behavioural assays to identify a novel subset of DA neurons that regulate age-associated male courtship activity in Drosophila. We find that increasing DA levels in a subset of cells in the PPL2ab neuronal cluster is necessary and sufficient for increased sustained courtship in both young and aged male flies. Our results indicate that preventing the age-related decline in DA levels in PPL2ab neurons alleviates diminished courtship behaviours in male Drosophila. These results may provide the foundation for deciphering the circuitry involved in sexual motivation in the male Drosophila brain.

The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca(2+) influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca(2+) influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.

The GAL4/UAS gene expression system is a precise means of targeted gene expression employed to study biological phenomena in Drosophila. A modified GAL4/UAS system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (LexAop) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/UAS system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in Drosophila.

The nervous and endocrine systems coordinate with each other to closely influence physiological and behavioural responses in animals. Here we show that WAKE (encoded by wide awake, also known as wake) modulates membrane levels of GABAA receptor Resistance to Dieldrin (Rdl), in insulin-producing cells of adult male Drosophila melanogaster. This results in changes to secretion of insulin-like peptides which is associated with changes in juvenile hormone biosynthesis in the corpus allatum, which in turn leads to a decrease in 20-hydroxyecdysone levels. A reduction in ecdysone signalling changes neural architecture and lowers the perception of the male-specific sex pheromone 11-cis-vaccenyl acetate by odorant receptor 67d olfactory neurons. These finding explain why WAKE-deficient in Drosophila elicits significant male-male courtship behaviour.

The harmful impact of the heavy metal lead on human health has been known for years. However, materials that contain lead remain in the environment. Measuring the blood lead level (BLL) is the only way to officially evaluate the degree of exposure to lead. The so-called "safe value" of the BLL seems to unreliably represent the secure threshold for children. In general, lead's underlying toxicological mechanism remains unclear and needs to be elucidated. Therefore, we developed a novel genetically encoded fluorescence resonance energy transfer (FRET)-based lead biosensor, Met-lead, and applied it to transgenic Drosophila to perform further investigations. We combined Met-lead with the UAS-GAL4 system to the sensor protein specifically expressed within certain regions of fly brains. Using a suitable imaging platform, including a fast epifluorescent or confocal laser-scanning/two-photon microscope with high resolution, we recorded the changes in lead content inside fly brains ex vivo and in vivo and at different life stages. The blood-brain barrier was found to play an important role in the protection of neurons in the brain against damage due to the heavy metal lead, either through food or microinjection into the abdomen. Met-lead has the potential to be a powerful tool for the sensing of lead within living organisms by employing either a fast epi-FRET microscope or high-resolution brain imaging.

In order to identify genes involved in stress and metabolic regulation, we carried out a Drosophila P-element-mediated mutagenesis screen for starvation resistance. We isolated a mutant, m2, that showed a 23% increase in survival time under starvation conditions. The P-element insertion was mapped to the region upstream of the vha16-1 gene, which encodes the c subunit of the vacuolar-type H+-ATPase. We found that vha16-1 is highly expressed in the fly midgut, and that m2 mutant flies are hypomorphic for vha16-1 and also exhibit reduced midgut acidity. This deficit is likely to induce altered metabolism and contribute to accelerated aging, since vha16-1 mutant flies are short-lived and display increases in body weight and lipid accumulation. Similar phenotypes were also induced by pharmacological treatment, through feeding normal flies and mice with a carbonic anhydrase inhibitor (acetazolamide) or proton pump inhibitor (PPI, lansoprazole) to suppress gut acid production. Our study may thus provide a useful model for investigating chronic acid suppression in patients.

Molecular and electrophysiological properties of NMDARs suggest that they may be the Hebbian "coincidence detectors" hypothesized to underlie associative learning. Because of the nonspecificity of drugs that modulate NMDAR function or the relatively chronic genetic manipulations of various NMDAR subunits from mammalian studies, conclusive evidence for such an acute role for NMDARs in adult behavioral plasticity, however, is lacking. Moreover, a role for NMDARs in memory consolidation remains controversial.

The intake of water is important for the survival of all animals and drinking water can be used as a reward in thirsty animals. Here we found that thirsty Drosophila melanogaster can associate drinking water with an odour to form a protein-synthesis-dependent water-reward long-term memory (LTM). Furthermore, we found that the reinforcement of LTM requires water-responsive dopaminergic neurons projecting to the restricted region of mushroom body (MB) β' lobe, which are different from the neurons required for the reinforcement of learning and short-term memory (STM). Synaptic output from α'β' neurons is required for consolidation, whereas the output from γ and αβ neurons is required for the retrieval of LTM. Finally, two types of MB efferent neurons retrieve LTM from γ and αβ neurons by releasing glutamate and acetylcholine, respectively. Our results therefore cast light on the cellular and molecular mechanisms responsible for processing water-reward LTM in Drosophila.

Long-term memory (LTM) formation depends on the conversed cAMP response element-binding protein (CREB)-dependent gene transcription followed by de novo protein synthesis. Thirsty fruit flies can be trained to associate an odor with water reward to form water-reward LTM (wLTM), which can last for over 24 hours without a significant decline. The role of de novo protein synthesis and CREB-regulated gene expression changes in neural circuits that contribute to wLTM remains unclear. Here, we show that acute inhibition of protein synthesis in the mushroom body (MB) αβ or γ neurons during memory formation using a cold-sensitive ribosome-inactivating toxin disrupts wLTM. Furthermore, adult stage-specific expression of dCREB2b in αβ or γ neurons also disrupts wLTM. The MB αβ and γ neurons can be further classified into five different neuronal subsets including αβ core, αβ surface, αβ posterior, γ main, and γ dorsal. We observed that the neurotransmission from αβ surface and γ dorsal neuron subsets is required for wLTM retrieval, whereas the αβ core, αβ posterior, and γ main are dispensable. Adult stage-specific expression of dCREB2b in αβ surface and γ dorsal neurons inhibits wLTM formation. In vivo calcium imaging revealed that αβ surface and γ dorsal neurons form wLTM traces with different dynamic properties, and these memory traces are abolished by dCREB2b expression. Our results suggest that a small population of neurons within the MB circuits support long-term storage of water-reward memory in Drosophila.

Memory consolidation is a time-dependent process through which an unstable learned experience is transformed into a stable long-term memory; however, the circuit and molecular mechanisms underlying this process are poorly understood. The Drosophila mushroom body (MB) is a huge brain neuropil that plays a crucial role in olfactory memory. The MB neurons can be generally classified into three subsets: γ, αβ, and α'β'. Here, we report that water-reward long-term memory (wLTM) consolidation requires activity from α'β'-related mushroom body output neurons (MBONs) in a specific time window. wLTM consolidation requires neurotransmission in MBON-γ3β'1 during the 0-2 h period after training, and neurotransmission in MBON-α'2 is required during the 2-4 h period after training. Moreover, neurotransmission in MBON-α'1α'3 is required during the 0-4 h period after training. Intriguingly, blocking neurotransmission during consolidation or inhibiting serotonin biosynthesis in serotoninergic dorsal paired medial (DPM) neurons also disrupted the wLTM, suggesting that wLTM consolidation requires serotonin signals from DPM neurons. The GFP Reconstitution Across Synaptic Partners (GRASP) data showed the connectivity between DPM neurons and MBON-γ3β'1, MBON-α'2, and MBON-α'1α'3, and RNAi-mediated silencing of serotonin receptors in MBON-γ3β'1, MBON-α'2, or MBON-α'1α'3 disrupted wLTM. Taken together, our results suggest that serotonin released from DPM neurons modulates neuronal activity in MBON-γ3β'1, MBON-α'2, and MBON-α'1α'3 at specific time windows, which is critical for the consolidation of wLTM in Drosophila.