Mushroom Body Research Articles

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.

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

BackgroundIn 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 methodWe 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. ResultsThis 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 methodsPrevious 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. ConclusionsThe embedding protocol for micro-computed tomography is suitable for calculating volume of the mushroom bodies in honey bee brains.

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.

Single-cell transcriptomes provide insights into expansion of glial cells in Bombyx mori.

The diversity of cell types in the brain and how these change during different developmental stages, remains largely unknown. The life cycle of insects is short and goes through 4 distinct stages including embryonic, larval, pupal, and adult stages. During postembryonic life, the larval brain transforms into a mature adult version after metamorphosis. The silkworm, Bombyx mori, is a lepidopteran model insect. Here, we characterized the brain cell repertoire of larval and adult B. mori by obtaining 50708 single-cell transcriptomes. Seventeen and 12 cell clusters from larval and adult brains were assigned based on marker genes, respectively. Identified cell types include Kenyon cells, optic lobe cells, monoaminergic neurons, surface glia, and astrocyte glia. We further assessed the cell type compositions of larval and adult brains. We found that the transition from larva to adult resulted in great expansion of glial cells. The glial cell accounted for 49.8% of adult midbrain cells. Compared to flies and ants, the mushroom body kenyon cell is insufficient in B. mori, which accounts for 5.4% and 3.6% in larval and adult brains, respectively. Analysis of neuropeptide expression showed that the abundance and specificity of expression varied among individual neuropeptides. Intriguingly, we found that ion transport peptide was specifically expressed in glial cells of larval and adult brains. The cell atlas dataset provides an important resource to explore cell diversity, neural circuits and genetic profiles.

Comparative neuroanatomy of the central nervous system in web-building and cursorial hunting spiders.

Spiders (Araneae) include cursorial species that stalk their prey and more stationary species that use webs for prey capture. While many cursorial hunting spiders rely on visual cues, web-building spiders use vibratory cues (mechanosensation) for prey capture. We predicted that the differences in primary sensory input between the species are mirrored by differences in the morphology/architecture of the central nervous system (CNS). Here, we investigated the CNS anatomy of four spider species, two cursorial hunters Pardosa amentata (Lycosidae) and Marpissa muscosa (Salticidae), and two web-building hunters Argiope bruennichi (Araneidae) and Parasteatoda tepidariorum (Theridiidae). Their CNS was analyzed using Bodian silver impregnations, immunohistochemistry, and microCT analysis. We found that there are major differences between species in the secondary eye pathway of the brain that pertain to first-order, second-order, and higher order brain centers (mushroom bodies [MB]). While P. amentata and M. muscosa have prominent visual neuropils and MB, these are much reduced in the two web-building species. Argiope bruennichi lacks second-order visual neuropils but has specialized photoreceptors that project into two distinct visual neuropils, and P. tepidariorum lacks MB, suggesting that motion vision might be absent in this species. Interestingly, the differences in the ventral nerve cord are much less pronounced, but the web-building spiders have proportionally larger leg neuropils than the cursorial spiders. Our findings suggest that the importance of visual information is much reduced in web-building spiders, compared to cursorial spiders, while processing of mechanosensory information requires the same major circuits in both web-building and cursorial hunting spiders.

Effects of stochastic coding on olfactory discrimination in flies and mice.

Sparse coding can improve discrimination of sensory stimuli by reducing overlap between their representations. Two factors, however, can offset sparse coding's benefits: similar sensory stimuli have significant overlap and responses vary across trials. To elucidate the effects of these 2 factors, we analyzed odor responses in the fly and mouse olfactory regions implicated in learning and discrimination-the mushroom body (MB) and the piriform cortex (PCx). We found that neuronal responses fall along a continuum from extremely reliable across trials to extremely variable or stochastic. Computationally, we show that the observed variability arises from noise within central circuits rather than sensory noise. We propose this coding scheme to be advantageous for coarse- and fine-odor discrimination. More reliable cells enable quick discrimination between dissimilar odors. For similar odors, however, these cells overlap and do not provide distinguishing information. By contrast, more unreliable cells are decorrelated for similar odors, providing distinguishing information, though these benefits only accrue with extended training with more trials. Overall, we have uncovered a conserved, stochastic coding scheme in vertebrates and invertebrates, and we identify a candidate mechanism, based on variability in a winner-take-all (WTA) inhibitory circuit, that improves discrimination with training.

Differentiation of workers into soldiers is associated with a size reduction of higher-order brain centers in the neotropical termite Procornitermes araujoi

Comparing the size of functionally distinct brain regions across individuals with remarkable differences in sensory processing and cognitive demands provides important insights into the selective forces shaping animal nervous systems. We took advantage of the complex system of worker-to-soldier differentiation in the termitid Procornitermes araujoi, to investigate how a profound modification of body morphology followed by an irreversible shift in task performance are translated in terms of brain structure and size. This behavioural shift is characterised by a reduction of the once wide and complex behavioural repertoire of workers to one exclusively dedicated to nest defence (soldiers). In accordance with soldier’s reduced cognitive and sensory demands, we show here that differentiation of workers into soldiers is associated with a size reduction of the mushroom body (MB) compartments, higher-order brain regions responsible for multimodal processing and integration of sensory information, as well as learning, memory, and decision-making. Moreover, in soldiers, we found an apparent fusion of the medial and lateral MB calyces likely associated with its volume reduction. These results illustrate a functional neuroplasticity of the MB associated with division of labour, supporting the link between MB size and behavioural flexibility in social insect workers.

Modelling TDP-43 proteinopathy in Drosophila uncovers shared and neuron-specific targets across ALS and FTD relevant circuits

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comprise a spectrum of neurodegenerative diseases linked to TDP-43 proteinopathy, which at the cellular level, is characterized by loss of nuclear TDP-43 and accumulation of cytoplasmic TDP-43 inclusions that ultimately cause RNA processing defects including dysregulation of splicing, mRNA transport and translation. Complementing our previous work in motor neurons, here we report a novel model of TDP-43 proteinopathy based on overexpression of TDP-43 in a subset of Drosophila Kenyon cells of the mushroom body (MB), a circuit with structural characteristics reminiscent of vertebrate cortical networks. This model recapitulates several aspects of dementia-relevant pathological features including age-dependent neuronal loss, nuclear depletion and cytoplasmic accumulation of TDP-43, and behavioral deficits in working memory and sleep that occur prior to axonal degeneration. RNA immunoprecipitations identify several candidate mRNA targets of TDP-43 in MBs, some of which are unique to the MB circuit and others that are shared with motor neurons. Among the latter is the glypican Dally-like-protein (Dlp), which exhibits significant TDP-43 associated reduction in expression during aging. Using genetic interactions we show that overexpression of Dlp in MBs mitigates TDP-43 dependent working memory deficits, conistent with Dlp acting as a mediator of TDP-43 toxicity. Substantiating our findings in the fly model, we find that the expression of GPC6 mRNA, a human ortholog of dlp, is specifically altered in neurons exhibiting the molecular signature of TDP-43 pathology in FTD patient brains. These findings suggest that circuit-specific Drosophila models provide a platform for uncovering shared or disease-specific molecular mechanisms and vulnerabilities across the spectrum of TDP-43 proteinopathies.

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'β' mushroom body neurons (MBns), with weak activity in the sated state and strong activity in the hungry state. Furthermore, we showed that α'β' MBn receives the same level of hot input from the mALT projection neurons via cholinergic transmission in sated and hungry states. Differences in α'β' 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'β' MBn to regulate HAB in different feeding conditions. Finally, we showed that the 2 α'β'-related MB output neurons (MBONs), MBON-α'3 and MBON-β'1, are necessary for the output of integrated hot avoidance information from α'β' MBn. Our results demonstrate the presence of dual insulin modulation pathways in α'β' MBn, which are important for suitable behavioral responses in Drosophila during thermoregulation under different feeding states.

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.

Adult neurogenesis does not explain the extensive post-eclosion growth of Heliconius mushroom bodies.

Among butterflies, Heliconius have a unique behavioural profile, being the sole genus to actively feed on pollen. Heliconius learn the location of pollen resources, and have enhanced visual memories and expanded mushroom bodies, an insect learning and memory centre, relative to related genera. These structures also show extensive post-eclosion growth and developmental sensitivity to environmental conditions. However, whether this reflects plasticity in neurite growth, or an extension of neurogenesis into the adult stage, is unknown. Adult neurogenesis has been described in some Lepidoptera, and could provide one route to the increased neuron number observed in Heliconius. Here, we compare volumetric changes in the mushroom bodies of freshly eclosed and aged Heliconius erato and Dryas iulia, and estimate the number of intrinsic mushroom body neurons using a new and validated automated method to count nuclei. Despite extensive volumetric variation associated with age, our data show that neuron number is remarkably constant in both species, suggesting a lack of adult neurogenesis in the mushroom bodies. We support this conclusion with assays of mitotic cells, which reveal very low levels of post-eclosion cell division. Our analyses provide an insight into the evolution of neural plasticity, and can serve as a basis for continued exploration of the potential mechanisms behind brain development and maturation.

Dopamine-Dependent Plasticity Is Heterogeneously Expressed by Presynaptic Calcium Activity across Individual Boutons of the Drosophila Mushroom Body.

The Drosophila mushroom body (MB) is an important model system for studying the synaptic mechanisms of associative learning. In this system, coincidence of odor-evoked calcium influx and dopaminergic input in the presynaptic terminals of Kenyon cells (KCs), the principal neurons of the MB, triggers long-term depression (LTD), which plays a critical role in olfactory learning. However, it is controversial whether such synaptic plasticity is accompanied by a corresponding decrease in odor-evoked calcium activity in the KC presynaptic terminals. Here, we address this question by inducing LTD by pairing odor presentation with optogenetic activation of dopaminergic neurons (DANs). This allows us to rigorously compare the changes at the presynaptic and postsynaptic sites in the same conditions. By imaging presynaptic acetylcholine release in the condition where LTD is reliably observed in the postsynaptic calcium signals, we show that neurotransmitter release from KCs is depressed selectively in the MB compartments innervated by activated DANs, demonstrating the presynaptic nature of LTD. However, total odor-evoked calcium activity of the KC axon bundles does not show concurrent depression. We further conduct calcium imaging in individual presynaptic boutons and uncover the highly heterogeneous nature of calcium plasticity. Namely, only a subset of boutons, which are strongly activated by associated odors, undergo calcium activity depression, while weakly responding boutons show potentiation. Thus, our results suggest an unexpected nonlinear relationship between presynaptic calcium influx and the results of plasticity, challenging the simple view of cooperative actions of presynaptic calcium and dopaminergic input.

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.

An antagonism between Spinophilin and Syd-1 operates upstream of memory-promoting presynaptic long-term plasticity.

We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here, we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila. While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.

Chemokine-like Orion is involved in the transformation of glial cells into phagocytes in different developmental neuronal remodeling paradigms.

During animal development, neurons often form exuberant or inappropriate axons and dendrites at early stages, followed by the refinement of neuronal circuits at late stages. Neural circuit refinement leads to the production of neuronal debris in the form of neuronal cell corpses, fragmented axons and dendrites, and pruned synapses requiring disposal. Glial cells act as predominant phagocytes during neuronal remodeling and degeneration, and critical signaling pathways between neurons and glia are necessary for the execution of phagocytosis. Chemokine-like mushroom body neuron-secreted Orion is essential for astrocyte infiltration into the γ axon bundle leading to γ axon pruning. Here we show a role of orion in debris engulfment and phagocytosis. Interestingly, orion is involved in the overall transformation of astrocytes into phagocytes. In addition, analysis of several neuronal paradigms demonstrates the role of orion in eliminating both peptidergic vCrz+ and PDF-Tri neurons via additional phagocytic glial cells like cortex and/or ensheathing glia. Our results suggest that Orion is essential for phagocytic activation of astrocytes, cortex, and ensheathing glia, and point to Orion as a trigger of glial infiltration, engulfment and phagocytosis.

Possible functions of ecdysone signaling reiteratively used in the adult honey bee brain

The European honey bee is a model organism for investigating the molecular and neural bases of the brain underlying social behaviors. Mushroom bodies (MBs) are a higher-order center of memory, learning, and sensory integration in insect brains, and honey bee MBs are a model to study adult neuronal plasticity. In the honey bee, MBs comprise three Class I Kenyon cell (KC) subtypes: large-, middle-, and small-type KCs, which are distinguished based on the size and localization of their somata, and gene expression profiles. One of the unique characteristics of honey bee MBs is that genes for ecdysone signaling are expressed in a spatially and temporarily regulated manner in the adult brain, suggesting that they play a role in the functional specialization of each KC subtype and behavioral control. A recent study reported that the transcription factor Mblk-1/E93, which functions downstream of ecdysone signaling during metamorphosis, targets genes involved in synaptic plasticity underlying memory and learning ability in the adult honey bee brain. On the other hand, the ecdysone receptor (EcR), which is expressed in small-type KCs in the MBs, was reported to target genes involved in lipid metabolism in the brain during foraging flight. The target genes for Mblk-1 and EcR in the adult brains differed from those during metamorphosis, implying that the reiterative use of some transcription factors involved in ecdysone signaling, such as EcR and Mblk-1, has contributed to the acquisition of novel MB functions in Aculeata species, including the honey bee.

Dopaminergic neurons dynamically update sensory values during olfactory maneuver

Dopaminergic neurons (DANs) drive associative learning to update the value of sensory cues, but their contribution to the assessment of sensory values outside the context of association remains largely unexplored. Here, we show in Drosophila that DANs in the mushroom body encode the innate value of odors and constantly update the current value by inducing plasticity during olfactory maneuver. Our connectome-based network model linking all the way from the olfactory neurons to DANs reproduces the characteristics of DAN responses, proposing a concrete circuit mechanism for computation. Downstream of DANs, odors alone induce value- and dopamine-dependent changes in the activity of mushroom body output neurons, which store the current value of odors. Consistent with this neural plasticity, specific sets of DANs bidirectionally modulate flies' steering in a virtual olfactory environment. Thus, the DAN circuit known for discrete, associative learning also continuously updates odor values in a nonassociative manner.

Optogenetic induction of appetitive and aversive taste memories in Drosophila.

Tastes typically evoke innate behavioral responses that can be broadly categorized as acceptance or rejection. However, research in Drosophila melanogaster indicates that taste responses also exhibit plasticity through experience-dependent changes in mushroom body circuits. In this study, we develop a novel taste learning paradigm using closed-loop optogenetics. We find that appetitive and aversive taste memories can be formed by pairing gustatory stimuli with optogenetic activation of sensory neurons or dopaminergic neurons encoding reward or punishment. As with olfactory memories, distinct dopaminergic subpopulations drive the parallel formation of short- and long-term appetitive memories. Long-term memories are protein synthesis-dependent and have energetic requirements that are satisfied by a variety of caloric food sources or by direct stimulation of MB-MP1 dopaminergic neurons. Our paradigm affords new opportunities to probe plasticity mechanisms within the taste system and understand the extent to which taste responses depend on experience.

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.

Transcriptome analysis reveals the mechanism of polyethylene atmosphere packaging delaying browning of postharvest Pleurotus eryngii

To elucidate the mechanism of low-density polyethylene(LDPE) atmosphere packaging on delaying browning of Pleurotus eryngii under a postharvest low-temperature storage, this study focused on exploring the effects of two thicknesses (0.02 and 0.05 mm) LDPE packaging on some antioxidant enzymes, and stress markers in P. eryngii. The results showed that LDPE atmosphere packaging maintained the phenolic content and higher superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) levels, reduced malondialdehyde (MDA) content, electrolyte leakage rate and polyphenol oxidase (PPO) levels in the mushroom. Also, transcriptome analyses showed that LDPE atmosphere packaging regulated the expression of enzymes PPO, POD, CAT and APX-related genes, inhibited the expression of lipoxygenases (LOX) and phospholipase D (PLD)-related genes and regulated the l-glutathione (GSH) antioxidant system. Overall, LDPE atmosphere packaging delayed the reduction of unsaturated fatty acids and energy loss by maintaining the integrity of cell membranes and regulating important genes involved in membrane lipid metabolism, energy metabolism, and respiratory metabolism of P. eryngii. In addition, it activated the expression of genes related to GSH synthesis and metabolism, thus enhancing the antioxidant capacity of mushroom body, reducing the accumulation of ROS and delaying browning.