PKCδ is an activator of neuronal mitochondrial metabolism that mediates the spacing effect on memory consolidation.

Relevance-based selectivity and high energy cost are two distinct features of long-term memory (LTM) formation that warrant its default inhibition. Spaced repetition of learning is a highly conserved cognitive mechanism that can lift this inhibition. Here, we questioned how the spacing effect integrates experience selection and energy efficiency at the cellular and molecular levels. We showed in Drosophila that spaced training triggers LTM formation by extending over several hours an increased mitochondrial metabolic activity in neurons of the associative memory center, the mushroom bodies (MBs). We found that this effect is mediated by PKCδ, a member of the so-called 'novel PKC' family of enzymes, which uncovers the critical function of PKCδ in neurons as a regulator of mitochondrial metabolism for LTM. Additionally, PKCδ activation and translocation to mitochondria result from LTM-specific dopamine signaling on MB neurons. By bridging experience-dependent neuronal circuit activity with metabolic modulation of memory-encoding neurons, PKCδ signaling binds the cognitive and metabolic constraints underlying LTM formation into a unified gating mechanism.

Glial metabolism versatility regulates mushroom body-driven behavioral output in Drosophila.

Providing metabolic support to neurons is now recognized as a major function of glial cells that is conserved from invertebrates to vertebrates. However, research in this field has focused for more than two decades on the relevance of lactate and glial glycolysis for neuronal energy metabolism, while overlooking many other facets of glial metabolism and their impact on neuronal physiology, circuit activity, and behavior. Here, we review recent work that has unveiled new features of glial metabolism, especially in Drosophila, in the modulation of behavioral traits involving the mushroom bodies (MBs). These recent findings reveal that spatially and biochemically distinct modes of glucose-derived neuronal fueling are implemented within the MB in a memory type-specific manner. In addition, cortex glia are endowed with several antioxidant functions, whereas astrocytes can serve as pro-oxidant agents that are beneficial to redox signaling underlying long-term memory. Finally, glial fatty acid oxidation seems to play a dual fail-safe role: first, as a mode of energy production upon glucose shortage, and, second, as a factor underlying the clearance of excessive oxidative load during sleep. Altogether, these integrated studies performed in Drosophila indicate that glial metabolism has a deterministic role on behavior.

Spaced training activates Miro/Milton-dependent mitochondrial dynamics in neuronal axons to sustain long-term memory.

Neurons have differential and fluctuating energy needs across distinct cellular compartments, shaped by brain electrochemical activity associated with cognition. In vitro studies show that mitochondria transport from soma to axons is key to maintaining neuronal energy homeostasis. Nevertheless, whether the spatial distribution of neuronal mitochondria is dynamically adjusted in vivo in an experience-dependent manner remains unknown. In Drosophila, associative long-term memory (LTM) formation is initiated by an early and persistent upregulation of mitochondrial pyruvate flux in the axonal compartment of neurons in the mushroom body (MB). Through behavior experiments, super-resolution analysis of mitochondria morphology in the neuronal soma and in vivo mitochondrial fluorescence recovery after photobleaching (FRAP) measurements in the axons, we show that LTM induction, contrary to shorter-lived memories, is sustained by the departure of some mitochondria from MB neuronal soma and increased mitochondrial dynamics in the axonal compartment. Accordingly, impairing mitochondrial dynamics abolished the increased pyruvate consumption, specifically after spaced training and in the MB axonal compartment, thereby preventing LTM formation. Our results thus promote reorganization of the mitochondrial network in neurons as an integral step in elaborating high-order cognitive processes.

Glycolysis-derived alanine from glia fuels neuronal mitochondria for memory in Drosophila.

Glucose is the primary source of energy for the brain; however, it remains controversial whether, upon neuronal activation, glucose is primarily used by neurons for ATP production or if it is partially oxidized in astrocytes, as proposed by the astrocyte-neuron lactate shuttle model for glutamatergic neurons. Thus, an in vivo picture of glucose metabolism during cognitive processes is missing. Here, we uncover in Drosophila melanogaster a glia-to-neuron alanine transfer involving alanine aminotransferase that sustains memory formation. Following associative conditioning, glycolysis in glial cells produces alanine, which is back-converted into pyruvate in cholinergic neurons of the olfactory memory center to uphold their increased mitochondrial needs. Alanine, as a mediator of glia-neuron coupling, could be an alternative to lactate in cholinergic systems. In parallel, a dedicated glial glucose transporter imports glucose specifically for long-term memory, by directly transferring it to neurons for use by the pentose phosphate pathway. Our results demonstrate in vivo the compartmentalization of glucose metabolism between neurons and glial cells during memory formation.

A New Behavioral Paradigm for Visual Classical Conditioning in Drosophila.

Visual learning in animals is a remarkable cognitive ability that plays a crucial role in their survival and adaptation. Therefore, the ability to learn is highly conserved among animals. Despite lacking a centralized nervous system like vertebrates, invertebrates have demonstrated remarkable learning abilities. Here, we describe a simple behavioral assay that allows the analysis of visual associative learning in individually traceable freely walking adult fruit flies. The setup is based on the simple and widely used behavioral assay to study orientation behavior in flies. A single wing-clipped fly that has been starved for 21 h is placed on a platform where two unreachable opposite visual sets are displayed. This visual learning protocol was initially developed to study the cognitive ability of fruit flies to process numerical information. Through the application of the protocol, flies are able to associate a specific visual set with an appetitive reward. This association is revealed 2 h later during the testing session where we observed a change in their preference upon learning (i.e., change in their spontaneous preference). Moreover, this protocol could potentially be used to associate any other visual object/property to the reward, expanding the opportunities of studying visual learning in freely walking fruit flies at individual level.

Numerical discrimination in Drosophila melanogaster.

Sensitivity to numbers is a crucial cognitive ability. The lack of experimental models amenable to systematic genetic and neural manipulation has precluded discovering neural circuits required for numerical cognition. Here, we demonstrate that Drosophila flies spontaneously prefer sets containing larger numbers of objects. This preference is determined by the ratio between the two numerical quantities tested, a characteristic signature of numerical cognition across species. Individual flies maintained their numerical choice over consecutive days. Using a numerical visual conditioning paradigm, we found that flies are capable of associating sucrose with numerical quantities and can be trained to reverse their spontaneous preference for large quantities. Finally, we show that silencing lobula columnar neurons (LC11) reduces the preference for more objects, thus identifying a neuronal substrate for numerical cognition in invertebrates. This discovery paves the way for the systematic analysis of the behavioral and neural mechanisms underlying the evolutionary conserved sensitivity to numerosity.

Developmental transcriptional control of mitochondrial homeostasis is required for activity-dependent synaptic connectivity.

During neuronal circuit formation, local control of axonal organelles ensures proper synaptic connectivity. Whether this process is genetically encoded is unclear and if so, its developmental regulatory mechanisms remain to be identified. We hypothesized that developmental transcription factors regulate critical parameters of organelle homeostasis that contribute to circuit wiring. We combined cell type-specific transcriptomics with a genetic screen to discover such factors. We identified Telomeric Zinc finger-Associated Protein (TZAP) as a temporal developmental regulator of neuronal mitochondrial homeostasis genes, including Pink1 . In Drosophila , loss of dTzap function during visual circuit development leads to loss of activity-dependent synaptic connectivity, that can be rescued by Pink1 expression. At the cellular level, loss of dTzap/TZAP leads to defects in mitochondrial morphology, attenuated calcium uptake and reduced synaptic vesicle release in fly and mammalian neurons. Our findings highlight developmental transcriptional regulation of mitochondrial homeostasis as a key factor in activity-dependent synaptic connectivity.

The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance.

FTSJ1 is a conserved human 2'-O-methyltransferase (Nm-MTase) that modifies several tRNAs at position 32 and the wobble position 34 in the anticodon loop. Its loss of function has been linked to X-linked intellectual disability (XLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here, we report a novel FTSJ1 pathogenic variant from an X-linked intellectual disability patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation on all human tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neural progenitor cells into neurons displays long and thin spine neurites compared with control cells. These defects are also observed in Drosophila and are associated with long-term memory deficits. Altogether, our study adds insight into FTSJ1 pathologies in humans and flies by the identification of novel FTSJ1 targets and the defect in neuron morphology.

Glia fuel neurons with locally synthesized ketone bodies to sustain memory under starvation.

During starvation, mammalian brains can adapt their metabolism, switching from glucose to alternative peripheral fuel sources. In the Drosophila starved brain, memory formation is subject to adaptative plasticity, but whether this adaptive plasticity relies on metabolic adaptation remains unclear. Here we show that during starvation, neurons of the fly olfactory memory centre import and use ketone bodies (KBs) as an energy substrate to sustain aversive memory formation. We identify local providers within the brain, the cortex glia, that use their own lipid store to synthesize KBs before exporting them to neurons via monocarboxylate transporters. Finally, we show that the master energy sensor AMP-activated protein kinase regulates both lipid mobilization and KB export in cortex glia. Our data provide a general schema of the metabolic interactions within the brain to support memory when glucose is scarce.

Glial glucose fuels the neuronal pentose phosphate pathway for long-term memory.

Brain function relies almost solely on glucose as an energy substrate. The main model of brain metabolism proposes that glucose is taken up and converted into lactate by astrocytes to fuel the energy-demanding neuronal activity underlying plasticity and memory. Whether direct neuronal glucose uptake is required for memory formation remains elusive. We uncover, in Drosophila, a mechanism of glucose shuttling to neurons from cortex glia, an exclusively perisomatic glial subtype, upon formation of olfactory long-term memory (LTM). In vivo imaging reveals that, downstream of cholinergic activation of cortex glia, autocrine insulin signaling increases glucose concentration in glia. Glucose is then transferred from glia to the neuronal somata in the olfactory memory center to fuel the pentose phosphate pathway and allow LTM formation. In contrast, our results indicate that the increase in neuronal glucose metabolism, although crucial for LTM formation, is not routed to glycolysis.

Enteric neurons increase maternal food intake during reproduction.

Reproduction induces increased food intake across females of many animal species1-4, providing a physiologically relevant paradigm for the exploration of appetite regulation. Here, by examining the diversity of enteric neurons in Drosophila melanogaster, we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. Our findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain.

Drosophila Middle-Term Memory: Amnesiac is Required for PKA Activation in the Mushroom Bodies, a Function Modulated by Neprilysin 1.

In Drosophila, the mushroom bodies (MB) constitute the central brain structure for olfactory associative memory. As in mammals, the cAMP/PKA pathway plays a key role in memory formation. In the MB, Rutabaga (Rut) adenylate cyclase acts as a coincidence detector during associative conditioning to integrate calcium influx resulting from acetylcholine stimulation and G-protein activation resulting from dopaminergic stimulation. Amnesiac encodes a secreted neuropeptide required in the MB for two phases of aversive olfactory memory. Previous sequence analysis has revealed strong homology with the mammalian pituitary adenylate cyclase-activating peptide (PACAP). Here, we examined whether amnesiac is involved in cAMP/PKA dynamics in response to dopamine and acetylcholine co-stimulation in living flies. Experiments were conducted with both sexes, or with either sex. Our data show that amnesiac is necessary for the PKA activation process that results from coincidence detection in the MB. Since PACAP peptide is cleaved by the human membrane neprilysin hNEP, we searched for an interaction between Amnesiac and Neprilysin 1 (Nep1), a fly neprilysin involved in memory. We show that when Nep1 expression is acutely knocked down in adult MB, memory deficits displayed by amn hypomorphic mutants are rescued. Consistently, Nep1 inhibition also restores normal PKA activation in amn mutant flies. Taken together, the results suggest that Nep1 targets Amnesiac degradation to terminate its signaling function. Our work thus highlights a key role for Amnesiac in establishing within the MB the PKA dynamics that sustain middle-term memory (MTM) formation, a function modulated by Nep1.SIGNIFICANCE STATEMENT The Drosophila amnesiac gene encodes a secreted neuropeptide whose expression is required for specific memory phases in the mushroom bodies (MB), the olfactory memory center. Here, we show that Amnesiac is required for PKA activation resulting from coincidence detection, a mechanism by which the MB integrate two spatially distinct stimuli to encode associative memory. Furthermore, our results uncover a functional relationship between Amnesiac and Neprilysin 1 (Nep1), a membrane peptidase involved in memory and expressed in the MB. These results suggest that Nep1 modulates Amnesiac levels. We propose that on conditioning, Amnesiac release from the MB allows, via an autocrine process, the sustaining of PKA activation-mediating memory, which subsequently is inactivated by Nep1 degradation.

In vivo large-scale analysis of Drosophila neuronal calcium traces by automated tracking of single somata.

How does the concerted activity of neuronal populations shape behavior? Impediments to address this question are primarily due to critical experimental barriers. An integrated perspective on large scale neural information processing requires an in vivo approach that can combine the advantages of exhaustively observing all neurons dedicated to a given type of stimulus, and simultaneously achieve a resolution that is precise enough to capture individual neuron activity. Current experimental data from in vivo observations are either restricted to a small fraction of the total number of neurons, or are based on larger brain volumes but at a low spatial and temporal resolution. Consequently, fundamental questions as to how sensory information is represented on a population scale remain unanswered. In Drosophila melanogaster, the mushroom body (MB) represents an excellent model to analyze sensory coding and memory plasticity. In this work, we present an experimental setup coupled with a dedicated computational method that provides in vivo measurements of the activity of hundreds of densely packed somata uniformly spread in the MB. We exploit spinning-disk confocal 3D imaging over time of the whole MB cell body layer in vivo while it is exposed to olfactory stimulation. Importantly, to derive individual signal from densely packed somata, we have developed a fully automated image analysis procedure that takes advantage of the specificities of our data. After anisotropy correction, our approach operates a dedicated spot detection and registration over the entire time sequence to transform trajectories to identifiable clusters. This enabled us to discard spurious detections and reconstruct missing ones in a robust way. We demonstrate that this approach outperformed existing methods in this specific context and made possible high-throughput analysis of approximately 500 single somata uniformly spread over the MB in various conditions. Applying this approach, we find that learned experiences change the population code of odor representations in the MB. After long-term memory (LTM) formation, we quantified an increase in responsive somata count and a stable single neuron signal. We predict that this method, which should further enable studying the population pattern of neuronal activity, has the potential to uncover fine details of sensory processing and memory plasticity.

Interactions between amyloid precursor protein-like (APPL) and MAGUK scaffolding proteins contribute to appetitive long-term memory in Drosophila melanogaster.

Amyloid precursor protein (APP), the precursor of amyloid beta peptide, plays a central role in Alzheimer's disease (AD), a pathology characterized by memory decline and synaptic loss upon aging. Understanding the physiological role of APP is fundamental in deciphering the progression of AD, and several studies suggest a synaptic function via protein-protein interactions. Nevertheless, it remains unclear whether and how these interactions contribute to memory. In Drosophila, we previously showed that APP-like (APPL), the fly APP homolog, is required for aversive associative memory in the olfactory memory center, the mushroom body (MB). In the present study, we show that APPL is required for appetitive long-term memory (LTM), another form of associative memory, in a specific neuronal subpopulation of the MB, the α'/ß' Kenyon cells. Using a biochemical approach, we identify the synaptic MAGUK (membrane-associated guanylate kinase) proteins X11, CASK, Dlgh2 and Dlgh4 as interactants of the APP intracellular domain (AICD). Next, we show that the Drosophila homologs CASK and Dlg are also required for appetitive LTM in the α'/ß' neurons. Finally, using a double RNAi approach, we demonstrate that genetic interactions between APPL and CASK, as well as between APPL and Dlg, are critical for appetitive LTM. In summary, our results suggest that APPL contributes to associative long-term memory through its interactions with the main synaptic scaffolding proteins CASK and Dlg. This function should be conserved across species.

"One Must Imagine Sisyphus Happy": Unveiling the Intimate Secrets of a Tenacious Fruitfly.

Perseverance in foraging is a high-risk/high-gain strategy. In this issue of Neuron, Sayin et al. (2019) decipher the neuronal circuit that arbitrates this choice in Drosophila. The fly's remarkable tenacity illuminates the interaction between working memory and decision making.

Sex Differences in Intestinal Carbohydrate Metabolism Promote Food Intake and Sperm Maturation.

Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. Here, we use the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. We find that carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. Our work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized.

Amnesiac Is Required in the Adult Mushroom Body for Memory Formation.

It was proposed that the Drosophila amnesiac gene (amn) is required for consolidation of aversive memory in the dorsal paired medial (DPM) neurons, a pair of large neurons that broadly innervate the mushroom bodies (MB), the fly center for olfactory learning and memory (Waddell et al., 2000). Yet, a conditional analysis showed that it was not possible to rescue the memory deficit of amnX8 null mutant flies when amn expression was restored only in the adult (DeZazzo et al., 1999), which led the authors to suggest that amn might be involved in the development of brain structures that normally promote adult olfactory memory. To further investigate temporal and spatial requirements of Amnesiac (AMN) peptide in memory, we used RNA interference in combination with conditional drivers. Experiments were conducted either in both sexes, or in either sexes. Our data show that acute modulation of amn expression in adult DPM neurons does not impact memory. We further show that amn expression is required for normal development of DPM neurons. Detailed enhancer trap analyses suggest that amn transcription unit contains two distinct enhancers, one specific of DPM neurons, and the other specific of α/ß MB neurons. This prompted us to investigate extensively the role of AMN in the adult MB. Together, our results demonstrate that amn is acutely required in adult α/ß MB neurons for middle-term and long-term memory. The data thus establish that amn plays two distinct roles. Its expression is required in DPM neurons for their development, and in adult MB for olfactory memory.SIGNIFICANCE STATEMENT The Drosophila amnesiac gene encodes a neuropeptide whose expression was proposed to be required for consolidation of aversive memory in the dorsal paired medial (DPM) neurons, a pair of large neurons that broadly innervate the mushroom bodies (MB), the olfactory memory center. Here, we investigated amnesiac temporal and spatial requirement using conditional tools that allowed us to manipulate its expression in selected neurons. This work leads to a complete reassessment of the role of amnesiac in brain development and memory. We show that amnesiac is required for two distinct processes: for normal development of DPM neurons, and in adult MB for memory.

Communication from Learned to Innate Olfactory Processing Centers Is Required for Memory Retrieval in Drosophila.

The behavioral response to a sensory stimulus may depend on both learned and innate neuronal representations. How these circuits interact to produce appropriate behavior is unknown. In Drosophila, the lateral horn (LH) and mushroom body (MB) are thought to mediate innate and learned olfactory behavior, respectively, although LH function has not been tested directly. Here we identify two LH cell types (PD2a1 and PD2b1) that receive input from an MB output neuron required for recall of aversive olfactory memories. These neurons are required for aversive memory retrieval and modulated by training. Connectomics data demonstrate that PD2a1 and PD2b1 neurons also receive direct input from food odor-encoding neurons. Consistent with this, PD2a1 and PD2b1 are also necessary for unlearned attraction to some odors, indicating that these neurons have a dual behavioral role. This provides a circuit mechanism by which learned and innate olfactory information can interact in identified neurons to produce appropriate behavior. VIDEO ABSTRACT.

A GABAergic Feedback Shapes Dopaminergic Input on the Drosophila Mushroom Body to Promote Appetitive Long-Term Memory.

Memory consolidation is a crucial step for long-term memory (LTM) storage. However, we still lack a clear picture of how memory consolidation is regulated at the neuronal circuit level. Here, we took advantage of the well-described anatomy of the Drosophila olfactory memory center, the mushroom body (MB), to address this question in the context of appetitive LTM. The MB lobes, which are made by the fascicled axons of the MB intrinsic neurons, are organized into discrete anatomical modules, each covered by the terminals of a defined type of dopaminergic neuron (DAN) and the dendrites of a corresponding type of MB output neuron (MBON). We previously revealed the essential role of one DAN, the MP1 neuron, in the formation of appetitive LTM. The MP1 neuron is anatomically matched to the GABAergic MBON MVP2, which has been attributed feedforward inhibitory functions recently. Here, we used behavior experiments and in vivo imaging to challenge the existence of MP1-MVP2 synapses and investigate their role in appetitive LTM consolidation. We show that MP1 and MVP2 neurons form an anatomically and functionally recurrent circuit, which features a feedback inhibition that regulates consolidation of appetitive memory. This circuit involves two opposite type 1 and type 2 dopamine receptors in MVP2 neurons and the metabotropic GABAB-R1 receptor in MP1 neurons. We propose that this dual-receptor feedback supports a bidirectional self-regulation of MP1 input to the MB. This mechanism displays striking similarities with the mammalian reward system, in which modulation of the dopaminergic signal is primarily assigned to inhibitory neurons.