Evolution of neural circuitry and cognition.

Neural circuits govern the interface between the external environment, internal cues and outwardly directed behaviours. To process multiple environmental stimuli and integrate these with internal state requires considerable neural computation. Expansion in neural network size, most readily represented by whole brain size, has historically been linked to behavioural complexity, or the predominance of cognitive behaviours. Yet, it is largely unclear which aspects of circuit variation impact variation in performance. A key question in the field of evolutionary neurobiology is therefore how neural circuits evolve to allow improved behavioural performance or innovation. We discuss this question by first exploring how volumetric changes in brain areas reflect actual neural circuit change. We explore three major axes of neural circuit evolution-replication, restructuring and reconditioning of cells and circuits-and discuss how these could relate to broader phenotypes and behavioural variation. This discussion touches on the relevant uses and limitations of volumetrics, while advocating a more circuit-based view of cognition. We then use this framework to showcase an example from the insect brain, the multi-sensory integration and internal processing that is shared between the mushroom bodies and central complex. We end by identifying future trends in this research area, which promise to advance the field of evolutionary neurobiology.

Learning with sparse reward in a gap junction network inspired by the insect mushroom body.

Animals can learn in real-life scenarios where rewards are often only available when a goal is achieved. This 'distal' or 'sparse' reward problem remains a challenge for conventional reinforcement learning algorithms. Here we investigate an algorithm for learning in such scenarios, inspired by the possibility that axo-axonal gap junction connections, observed in neural circuits with parallel fibres such as the insect mushroom body, could form a resistive network. In such a network, an active node represents the task state, connections between nodes represent state transitions and their connection to actions, and current flow to a target state can guide decision making. Building on evidence that gap junction weights are adaptive, we propose that experience of a task can modulate the connections to form a graph encoding the task structure. We demonstrate that the approach can be used for efficient reinforcement learning under sparse rewards, and discuss whether it is plausible as an account of the insect mushroom body.

The role of glial and neuronal Eph/ephrin signaling in Drosophila mushroom body development and sleep and circadian behavior.

The Eph receptor, a prototypically large receptor protein tyrosine kinase, interacts with ephrin ligands, forming a bidirectional signaling system that impacts diverse brain functions. Eph receptors and ephrins mediate forward and reverse signaling, affecting neurogenesis, axon guidance, and synaptic signaling. While mammalian studies have emphasized their roles in neurogenesis and synaptic plasticity, the Drosophila counterparts are less studied, especially in glial cells, despite structural similarities. Using RNAi to modulate Eph/ephrin expression in Drosophila neurons and glia, we studied their roles in brain development and sleep and circadian behavior. Knockdown of neuronal ephrin disrupted mushroom body development, while glial knockdown had minimal impact. Surprisingly, disrupting ephrin in neurons or glial cells altered sleep and circadian rhythms, indicating a direct involvement in these behaviors independent from developmental effects. Further analysis revealed distinct sleep phenotypes between neuronal and glial knockdowns, underscoring the intricate interplay within the neural circuits that govern behavior. Glia-specific knockdowns showed altered sleep patterns and reduced circadian rhythmicity, suggesting an intricate role of glia in sleep regulation. Our findings challenge simplistic models of Eph/ephrin signaling limited to neuron-glia communication and emphasize the complexity of the regulatory networks modulating behavior. Future investigations targeting specific glial subtypes will enhance our understanding of Eph/ephrin signaling's role in sleep regulation across species.

Mrj is a chaperone of the Hsp40 family that regulates Orb2 oligomerization and long-term memory in Drosophila.

Orb2 the Drosophila homolog of cytoplasmic polyadenylation element binding (CPEB) protein forms prion-like oligomers. These oligomers consist of Orb2A and Orb2B isoforms and their formation is dependent on the oligomerization of the Orb2A isoform. Drosophila with a mutation diminishing Orb2A's prion-like oligomerization forms long-term memory but fails to maintain it over time. Since this prion-like oligomerization of Orb2A plays a crucial role in the maintenance of memory, here, we aim to find what regulates this oligomerization. In an immunoprecipitation-based screen, we identify interactors of Orb2A in the Hsp40 and Hsp70 families of proteins. Among these, we find an Hsp40 family protein Mrj as a regulator of the conversion of Orb2A to its prion-like form. Mrj interacts with Hsp70 proteins and acts as a chaperone by interfering with the aggregation of pathogenic Huntingtin. Unlike its mammalian homolog, we find Drosophila Mrj is neither an essential gene nor causes any gross neurodevelopmental defect. We observe a loss of Mrj results in a reduction in Orb2 oligomers. Further, Mrj knockout exhibits a deficit in long-term memory and our observations suggest Mrj is needed in mushroom body neurons for the regulation of long-term memory. Our work implicates a chaperone Mrj in mechanisms of memory regulation through controlling the oligomerization of Orb2A and its association with the translating ribosomes.

Overexpression of the limk1 Gene in Drosophila melanogaster Can Lead to Suppression of Courtship Memory in Males.

Courtship suppression is a behavioral adaptation of the fruit fly. When majority of the females in a fly population are fertilized and non-receptive for mating, a male, after a series of failed attempts, decreases its courtship activity towards all females, saving its energy and reproductive resources. The time of courtship decrease depends on both duration of unsuccessful courtship and genetically determined features of the male nervous system. Thereby, courtship suppression paradigm can be used for studying molecular mechanisms of learning and memory. p-Cofilin, a component of the actin remodeling signaling cascade and product of LIM-kinase 1 (LIMK1), regulates Drosophila melanogaster forgetting in olfactory learning paradigm. Previously, we have shown that limk1 suppression in the specific types of nervous cells differently affects fly courtship memory. Here, we used Gal4 > UAS system to induce limk1 overexpression in the same types of neurons. limk1 activation in the mushroom body, glia, and fruitless neurons decreased learning index compared to the control strain or the strain with limk1 knockdown. In cholinergic and dopaminergic/serotoninergic neurons, both overexpression and knockdown of limk1 impaired Drosophila short-term memory. Thus, proper balance of the limk1 activity is crucial for normal cognitive activity of the fruit fly.

Tissue-specific O-GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning.

O-GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an O-GlcNAc binding activity derived from Clostridium perfringens OGA (CpOGA) with TurboID proximity labeling in Drosophila, we developed an O-GlcNAcylation profiling tool that translates O-GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the O-GlcNAc interactome in major brain regions of Drosophila and found that components of the translational machinery, particularly ribosomal subunits, were abundantly O-GlcNAcylated in the mushroom body of Drosophila brain. Hypo-O-GlcNAcylation induced by ectopic expression of active CpOGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of O-GlcNAcylation in Drosophila, and suggests a possibility that O-GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.

Newly synthesized proteins often receive further chemical modifications that change their structure and role in the cell. O-GlcNAcylation, for instance, consists in a certain type of sugar molecule being added onto dedicated protein segments. It is required for the central nervous system to develop and work properly; in fact, several neurological disorders such as Alzheimer's, Parkinson's or Huntington's disease are linked to disruptions in O-GlcNAcylation. However, scientists are currently lacking approaches that would allow them to reliably identify which proteins require O-GlcNAcylation in specific regions of the brain to ensure proper cognitive health. To address this gap, Yu et al. developed a profiling tool that allowed them to probe O-GlcNAcylation protein targets in different tissues of fruit flies. Their approach relies on genetically manipulating the animals so that a certain brain area overproduces two enzymes that work in tandem; the first binds specifically to O-GlcNAcylated proteins, which allows the second to add a small 'biotin' tag to them. Tagged proteins can then be captured and identified. Using this tool helped Yu et al. map out which proteins go through O-GlcNAcylation in various brain regions. This revealed, for example, that in the mushroom body ­ the 'learning center' of the fly brain ­ O-GlcNAcylation occurred predominantly in the protein-building machinery. To investigate the role of O-GlcNAcylation in protein synthesis and learning, Yu et al. used an approach that allowed them to decrease the levels of O-GlcNAcylation in the mushroom body. This resulted in reduced local protein production and the flies performing poorly in olfactory learning tasks. However, artificially increasing protein synthesis reversed these deficits. Overall, the work by Yu et al. provides a useful tool for studying the tissue-specific effects of O-GlcNAcylation in fruit flies, and its role in learning. Further studies should explore how this process may be linked to cognitive function by altering protein synthesis in the brain.

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.

Cyclic nucleotide-induced bidirectional long-term synaptic plasticity in Drosophila mushroom body.

Activation of the cAMP pathway is one of the common mechanisms underlying long-term potentiation (LTP). In the Drosophila mushroom body, simultaneous activation of odour-coding Kenyon cells (KCs) and reinforcement-coding dopaminergic neurons activates adenylyl cyclase in KC presynaptic terminals, which is believed to trigger synaptic plasticity underlying olfactory associative learning. However, learning induces long-term depression (LTD) at these synapses, contradicting the universal role of cAMP as a facilitator of transmission. Here, we developed a system to electrophysiologically monitor both short-term and long-term synaptic plasticity at KC output synapses and demonstrated that they are indeed an exception in which activation of the cAMP-protein kinase A pathway induces LTD. Contrary to the prevailing model, our cAMP imaging found no evidence for synergistic action of dopamine and KC activity on cAMP synthesis. Furthermore, we found that forskolin-induced cAMP increase alone was insufficient for plasticity induction; it additionally required simultaneous KC activation to replicate the presynaptic LTD induced by pairing with dopamine. On the other hand, activation of the cGMP pathway paired with KC activation induced slowly developing LTP, proving antagonistic actions of the two second-messenger pathways predicted by behavioural study. Finally, KC subtype-specific interrogation of synapses revealed that different KC subtypes exhibit distinct plasticity duration even among synapses on the same postsynaptic neuron. Thus, our work not only revises the role of cAMP in synaptic plasticity by uncovering the unexpected convergence point of the cAMP pathway and neuronal activity, but also establishes the methods to address physiological mechanisms of synaptic plasticity in this important model. KEY POINTS: Although presynaptic cAMP increase generally facilitates synapses, olfactory associative learning in Drosophila, which depends on dopamine and cAMP signalling genes, induces long-term depression (LTD) at the mushroom body output synapses. By combining electrophysiology, pharmacology and optogenetics, we directly demonstrate that these synapses are an exception where activation of the cAMP-protein kinase A pathway leads to presynaptic LTD. Dopamine- or forskolin-induced cAMP increase alone is not sufficient for LTD induction; neuronal activity, which has been believed to trigger cAMP synthesis in synergy with dopamine input, is required in the downstream pathway of cAMP. In contrast to cAMP, activation of the cGMP pathway paired with neuronal activity induces presynaptic long-term potentiation, which explains behaviourally observed opposing actions of transmitters co-released by dopaminergic neurons. Our work not only revises the role of cAMP in synaptic plasticity, but also provides essential methods to address physiological mechanisms of synaptic plasticity in this important model system.

Bisphenol F affects neurodevelopmental gene expression, mushroom body development, and behavior in Drosophila melanogaster.

Bisphenol F (BPF) is a potential neurotoxicant used as a replacement for bisphenol A (BPA) in polycarbonate plastics and epoxy resins. We investigated the neurodevelopmental impacts of BPF exposure using Drosophila melanogaster as a model. Our transcriptomic analysis indicated that developmental exposure to BPF caused the downregulation of neurodevelopmentally relevant genes, including those associated with synapse formation and neuronal projection. To investigate the functional outcome of BPF exposure, we evaluated neurodevelopmental impacts across two genetic strains of Drosophila- w1118 (control) and the Fragile X Syndrome (FXS) model-by examining both behavioral and neuronal phenotypes. We found that BPF exposure in w1118 Drosophila caused hypoactive larval locomotor activity, decreased time spent grooming by adults, reduced courtship activity, and increased the severity but not frequency of ß-lobe midline crossing defects by axons in the mushroom body. In contrast, although BPF reduced peristaltic contractions in FXS larvae, it had no impact on other larval locomotor phenotypes, grooming activity, or courtship activity. Strikingly, BPF exposure reduced both the severity and frequency of ß-lobe midline crossing defects in the mushroom body of FXS flies, a phenotype previously observed in FXS flies exposed to BPA. This data indicates that BPF can affect neurodevelopment and its impacts vary depending on genetic background. Further, BPF may elicit a gene-environment interaction with Drosophila fragile X messenger ribonucleoprotein 1 (dFmr1)-the ortholog of human FMR1, which causes fragile X syndrome and is the most common monogenetic cause of intellectual disability and autism spectrum disorder.

Asymmetric neurons are necessary for olfactory learning in the Drosophila brain.

Animals have complementary parallel memory systems that process signals from various sensory modalities. In the brain of the fruit fly Drosophila melanogaster, mushroom body (MB) circuitry is the primary associative neuropil, critical for all stages of olfactory memory. Here, our findings suggest that active signaling from specific asymmetric body (AB) neurons is also crucial for this process. These AB neurons respond to odors and electric shock separately and exhibit timing-sensitive neuronal activity in response to paired stimulation while leaving a decreased memory trace during retrieval. Our experiments also show that rutabaga-encoded adenylate cyclase, which mediates coincidence detection, is required for learning and short-term memory in both AB and MB. We observed additive effects when manipulating rutabaga co-expression in both structures. Together, these results implicate the AB in playing a critical role in associative olfactory learning and short-term memory.

Probiotic Lactobacillus spp. improves Drosophila memory by increasing lactate dehydrogenase levels in the brain mushroom body neurons.

Probiotics are live microorganisms that offer potential benefits to their hosts and can occasionally influence behavioral responses. However, the detailed mechanisms by which probiotics affect the behavior of their hosts and the underlying biogenic effects remain unclear. Lactic acid bacteria, specifically Lactobacillus spp. are known probiotics. Drosophila melanogaster, commonly known as the fruit fly, is a well-established model organism for investigating the interaction between the host and gut microbiota in translational research. Herein, we showed that 5-day administration of Lactobacillus acidophilus (termed GMNL-185) or Lacticaseibacillus rhamnosus (termed GMNL-680) enhances olfactory-associative memory in Drosophila. Moreover, a combined diet of GMNL-185 and GMNL-680 demonstrated synergistic effects on memory functions. Live brain imaging revealed a significant increase in calcium responses to the training odor in the mushroom body ß and γ lobes of flies that underwent mixed feeding with GMNL-185 and GMNL-680. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and whole-mount brain immunohistochemistry revealed significant upregulation of lactate dehydrogenase (LDH) expression in the fly brain following the mixed feeding. Notably, the genetic knockdown of Ldh in neurons, specifically in mushroom body, ameliorated the beneficial effects of mixed feeding with GMNL-185 and GMNL-680 on memory improvement. Altogether, our results demonstrate that supplementation with L. acidophilus and L. rhamnosus enhances memory functions in flies by increasing brain LDH levels.

New genetic tools for mushroom body output neurons in Drosophila.

How memories of past events influence behavior is a key question in neuroscience. The major associative learning center in Drosophila, the mushroom body (MB), communicates to the rest of the brain through mushroom body output neurons (MBONs). While 21 MBON cell types have their dendrites confined to small compartments of the MB lobes, analysis of EM connectomes revealed the presence of an additional 14 MBON cell types that are atypical in having dendritic input both within the MB lobes and in adjacent brain regions. Genetic reagents for manipulating atypical MBONs and experimental data on their functions have been lacking. In this report we describe new cell-type-specific GAL4 drivers for many MBONs, including the majority of atypical MBONs that extend the collection of MBON driver lines we have previously generated (Aso et al., 2014a; Aso et al., 2016; Aso et al., 2019). Using these genetic reagents, we conducted optogenetic activation screening to examine their ability to drive behaviors and learning. These reagents provide important new tools for the study of complex behaviors in Drosophila.

Neuroplasticity in honey bee brains: An enhanced micro-computed tomography protocol for precise mushroom body volume measurement.

BACKGROUND: In insect brains, mushroom bodies are associated with memory and learning behavior. It has been demonstrated that the volume of the mushroom bodies in the brain of a worker honey bee changes during the adult stage. Changes in mushroom body volume imply high neuroplasticity in the brains and may be related to the age polyethism of honey bees. A suitable volume measurement method is needed to understand the correlation between behavioral changes and mushroom body volume changes in honey bees. NEW METHOD: We developed a new protocol for insect micro-computed tomography by modifying a previously reported method. Permount™ mounting medium was used as the embedding medium for micro-computed tomography scanning. RESULTS: This protocol can generate images with high contrast inside the brain and reduce the marked shape changes during specimen processing. From the resulting high-contrast images, we used freeware to generate a three-dimensional model and calculate the volumes of the mushroom bodies in honey bees. The measured volumes of the mushroom bodies were larger than the values reported in most previous studies. There was no significant difference between the left and right mushroom body volumes, but the volumes of honey bee mushroom bodies significantly increased with age. COMPARISON WITH EXISTING METHODS: Previous protocols for micro-computed tomography using dried samples would cause brain shrinkage; protocols using ethanol-preserved or resin-embedded samples generated images with lower contrast. CONCLUSIONS: The embedding protocol for micro-computed tomography is suitable for calculating volume of the mushroom bodies in honey bee brains.

Glia transmit negative valence information during aversive learning in Drosophila.

During Drosophila aversive olfactory conditioning, aversive shock information needs to be transmitted to the mushroom bodies (MBs) to associate with odor information. We report that aversive information is transmitted by ensheathing glia (EG) that surround the MBs. Shock induces vesicular exocytosis of glutamate from EG. Blocking exocytosis impairs aversive learning, whereas activation of EG can replace aversive stimuli during conditioning. Glutamate released from EG binds to N-methyl-d-aspartate receptors in the MBs, but because of Mg2+ block, Ca2+ influx occurs only when flies are simultaneously exposed to an odor. Vesicular exocytosis from EG also induces shock-associated dopamine release, which plays a role in preventing formation of inappropriate associations. These results demonstrate that vesicular glutamate released from EG transmits negative valence information required for associative learning.

Transcriptomic responses to location learning by honeybee dancers are partly mirrored in the brains of dance-followers.

The waggle dances of honeybees are a strikingly complex form of animal communication that underlie the collective foraging behaviour of colonies. The mechanisms by which bees assess the locations of forage sites that they have visited for representation on the dancefloor are now well-understood, but few studies have considered the remarkable backward translation of such information into flight vectors by dance-followers. Here, we explore whether the gene expression patterns that are induced through individual learning about foraging locations are mirrored when bees learn about those same locations from their nest-mates. We first confirmed that the mushroom bodies of honeybee dancers show a specific transcriptomic response to learning about distance, and then showed that approximately 5% of those genes were also differentially expressed by bees that follow dances for the same foraging sites, but had never visited them. A subset of these genes were also differentially expressed when we manipulated distance perception through an optic flow paradigm, and responses to learning about target direction were also in part mirrored in the brains of dance followers. Our findings show a molecular footprint of the transfer of learnt information from one animal to another through this extraordinary communication system, highlighting the dynamic role of the genome in mediating even very short-term behavioural changes.

A modified method to analyse cell proliferation using EdU labelling in large insect brains.

The study of neurogenesis is critical to understanding of the evolution of nervous systems. Within invertebrates, this process has been extensively studied in Drosophila melanogaster, which is the predominant model thanks to the availability of advanced genetic tools. However, insect nervous systems are extremely diverse, and by studying a range of taxa we can gain additional information about how nervous systems and their development evolve. One example of the high diversity of insect nervous system diversity is provided by the mushroom bodies. Mushroom bodies have critical roles in learning and memory and vary dramatically across species in relative size and the type(s) of sensory information they process. Heliconiini butterflies provide a useful snapshot of this diversity within a closely related clade. Within Heliconiini, the genus Heliconius contains species where mushroom bodies are 3-4 times larger than other closely related genera, relative to the rest of the brain. This variation in size is largely explained by increases in the number of Kenyon cells, the intrinsic neurons which form the mushroom body. Hence, variation in mushroom body size is the product of changes in cell proliferation during Kenyon cell neurogenesis. Studying this variation requires adapting labelling techniques for use in less commonly studied organisms, as methods developed for common laboratory insects often do not work. Here, we present a modified protocol for EdU staining to examine neurogenesis in large-brained insects, using Heliconiini butterflies as our primary case, but also demonstrating applicability to cockroaches, another large-brained insect.

Mapping Kenyon cell inputs in Drosophila using dye electroporation.

Here, we describe a technique for charting the inputs of individual Kenyon cells in the Drosophila brain. In this technique, a single Kenyon cell per brain hemisphere is photo-labeled to visualize its claw-like dendritic terminals; a dye-filled electrode is used to backfill the projection neuron connected to each claw. This process can be repeated in hundreds of brains to build a connectivity matrix. Statistical analyses of such a matrix can reveal connectivity patterns such as random input and biased connectivity. For complete details on the use and execution of this protocol, please refer to Hayashi et al. (2022).1.

Decision-making: A new role for insect mushroom bodies.

Mushroom bodies are neural structures that are ubiquitous in insect brains, where they integrate sensory inputs to encode stimulus identity and meaning. A new study now adds action selection - decision-making - to those roles.

Impact of Drosophila LIM homeodomain protein Apterous on the morphology of the adult mushroom body.

A LIM homeodomain transcription factor Apterous (Ap) regulates embryonic and larval neurodevelopment in Drosophila. Although Ap is still expressed in the adult brain, it remains elusive whether Ap is involved in neurodevelopmental events in the adult brain because flies homozygous for ap mutations are usually lethal before they reach the adult stage. In this study, using adult escapers of ap knockout (KO) homozygotes, we examined whether the complete lack of ap expression affects the morphology of the mushroom body (MB) neurons and Pigment-dispersing factor (Pdf)-positive clock neurons in the adult brain. Although ap KO escapers showed severe structural defects of MB neurons, no clear morphological defects were found in Pdf-positive clock neurons. These results suggest that Ap in the adult brain is essential for the neurodevelopment of specific ap-positive neurons, but it is not necessarily involved in the development of all ap-positive neurons.

Independent insulin signaling modulators govern hot avoidance under different feeding states.

Thermosensation is critical for the survival of animals. However, mechanisms through which nutritional status modulates thermosensation remain unclear. Herein, we showed that hungry Drosophila exhibit a strong hot avoidance behavior (HAB) compared to food-sated flies. We identified that hot stimulus increases the activity of α'ß' mushroom body neurons (MBns), with weak activity in the sated state and strong activity in the hungry state. Furthermore, we showed that α'ß' MBn receives the same level of hot input from the mALT projection neurons via cholinergic transmission in sated and hungry states. Differences in α'ß' MBn activity between food-sated and hungry flies following heat stimuli are regulated by distinct Drosophila insulin-like peptides (Dilps). Dilp2 is secreted by insulin-producing cells (IPCs) and regulates HAB during satiety, whereas Dilp6 is secreted by the fat body and regulates HAB during the hungry state. We observed that Dilp2 induces PI3K/AKT signaling, whereas Dilp6 induces Ras/ERK signaling in α'ß' MBn to regulate HAB in different feeding conditions. Finally, we showed that the 2 α'ß'-related MB output neurons (MBONs), MBON-α'3 and MBON-ß'1, are necessary for the output of integrated hot avoidance information from α'ß' MBn. Our results demonstrate the presence of dual insulin modulation pathways in α'ß' MBn, which are important for suitable behavioral responses in Drosophila during thermoregulation under different feeding states.