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Mushroom Bodies

Mushroom Bodies are complex neuropil structures found in the brains of many arthropods, including insects and crustaceans.
They play a crucial role in learning, memory, and sensory integration.
These structures are composed of densely packed intrinsic neurons called Kenyon cells, which receive input from various sensory modalities and integrate this information to generate complex behaviors.
Mushroom Bodies are a fascianting area of research, with many unanswered questions about their precise functions and underlying neuronal circuitry.
Exploring these enigmatic brain regions can provide valuable insights into the evolution and diversity of invertebrate cognition.

Most cited protocols related to «Mushroom Bodies»

Neurons were traced in a serial section transmission electron microscopy (ssTEM) volume of a full adult female D. melanogaster brain (FAFB) (Zheng et al., 2018 (link)) using CATMAID, a web-based software for collaborative neural circuit reconstruction from large image datasets (https://catmaid.readthedocs.io/en/stable/) (Saalfeld et al., 2009 (link), Schneider-Mizell et al., 2016 ). Consistent with previous studies (Eichler et al., 2017 (link), Schlegel et al., 2016 , Schneider-Mizell et al., 2016 ), tracing followed the centerline of a neuron’s profiles through the dataset to reconstruct neurite morphology and annotate synaptic sites. We used an iterative approach established and tested by Schneider-Mizell et al. (2016) , where initial reconstruction is followed by a systematic proofreading by at least two experienced reviewers (> 500h of tracing experience).
MBON identification: MBONs were located by sampling downstream of previously identified KC synapses in the respective mushroom body lobe compartments. Their identity was confirmed by comparison with light level data (Aso et al., 2014b (link)).
Synapse annotation: Synaptic sites were identified based on three, previously described criteria (Prokop and Meinertzhagen, 2006 (link)) and reviewed as above: an active zone with (1) T-bar(s) and (2) surrounding vesicle cloud, and (3) a synaptic cleft to which all postsynaptic neurons must have access.
In Drosophila, presynapses have been found on fine axonal processes (Schneider-Mizell et al., 2016 ), boutons (Butcher et al., 2012 (link)), and other neurites that are neither in the dendritic nor the axonal field. Post-synapses have been found on large or fine dendritic processes and fine spine like twigs that are shorter than 3μm (Schneider-Mizell et al., 2016 ). M4β′ M6R and M6L were reconstructed the same way to maintain consistency in the placement of synapses. Schneider-Mizell et al. (2016) estimated that the tracing approach employed typically finds 99.8% of all pre- and 91.7% of all post-synapses. The probability of identifying false-positive post-synapses is 2.2% and negligible for presynapses. Since all synaptic sites on the MVP2 axon and M4β′ and M6 dendrites were annotated, we can estimate the upper and lower bounds of the number of synapses between MVP2 and M4β′ or M6 neurons (Note only integer numbers of synapses are expected):
FoundLower BoundUpper Bound
MVP2- > M6R1716.618.4
MVP2- > M6L1615.617.3
MVP2- > M4β'474650.8

Our error margins are likely smaller than those listed above, because the respective neurites of all neurons were more extensively reviewed than the agreed standard.
Reconstructed neurons were visualized using Blender 3D, an open-source 3D software (https://www.blender.org/). Neuron data from CATMAID were imported and shaded by Strahler order using an existing CATMAID plugin for Blender (https://github.com/schlegelp/CATMAID-to-Blender; Schlegel et al., 2016 ).
Volumetric reconstruction of synapse architecture was achieved by importing and annotating FAFB image data into ImageJ using the TrakEM2 plugin (Cardona et al., 2012 (link)). Reconstructions were exported for rendering to Blender 3D.
Analysis: All analyses were performed in R and Python using open-source software. PyMaid (https://github.com/schlegelp/PyMaid) and RCatmaid (http://jefferis.github.io/rcatmaid/; http://jefferis.github.io/elmr/) were used to interface with CATMAID servers and perform morphological analyses. Dendrogram representations of neural arbors were generated using new code (https://github.com/markuspleijzier/AdultEM/tree/master/Dendrogram_code) the graphviz library (https://graphviz.gitlab.io/; Gansner and North, 2000 via Python bindings provided by NetworkX, https://networkx.github.io/; Hagberg et al., 2008 ). M4β′ axonlets were defined as distal parts of neurites originating from the dendritic field, which made exclusively presynaptic connections. Axonlets were isolated and imported into Blender 3D using PyMaid. The root of the dendritic field was defined as the point at which the neuron’s main neurite branched into proximal dendrites and distal axon. Geodesic (along the arbor) distances between synapses and dendritic root were calculated using RCatmaid.
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Publication 2018
Axon Brain Dendrites DNA Library Drosophila Light Mitral Valve Prolapse, Myxomatous 2 Mushroom Bodies Nervousness Neurites Neurons Plant Roots Presynaptic Terminals Python Reconstructive Surgical Procedures Synapses Transmission, Communicable Disease Transmission Electron Microscopy Trees Vertebral Column Volume Electron Microscopy Woman
We used wild type (Oregon R) and white eyed (w1118) fruitflies, Drosophila melanogaster for immunocytochemistry and in situ hybridization. A number of Gal4 strains were used for driving GFP to identify specific sets of neurons in relation to sNPF markers. These were: gad1-Gal4 (Glutamic acid decarboxylase-1 promoter-Gal4 [81 (link)]; from G. Miesenböck, New Haven, CT), Cha-Gal4 (w*; P{Cha-GAL4.7.4}19B P{UAS-GFP.S65T}T2; choline acetyltransferase promotor-Gal4-GFP fusion, from Bloomington Drosophila Stock Center at Indiana University, IN [33 (link)]); th-Gal4 (tyrosine-hydroxylase promoter-Gal4; [38 (link)]; from S. Birmann, Marseille, France), tdc-Gal4 (tyrosine decarboxylase promoter Gal4; obtained from J. Dubnau, Cold Spring Harbor, NY; [82 (link)]), OK371-Gal4 (vesicular glutamate transporter-Gal4) (from H. Aberle, Tübingen, Germany [36 (link)]; OK107-Gal4 (P{GawB}OK107 (expression in mushroom body neurons); from Bloomington Stock center; [83 (link),84 (link)]), npf-Gal4 (Neuropeptide F-promoter-Gal4; from P. Shen, Athens, GA; [39 (link)]). A snpf-Gal4 (NP6301; order number 113901) was obtained from the Drosophila Genetic Resource Center (DGRC), Kyoto Institute of Technology, Kyoto, Japan. The genotype of this Gal4 is yw; P{GawB}NP6301/CyO, P{UAS-lacZ.UW14}UW14. To our knowledge the expression of this Gal4 has not been previously described. The enhancer trap line Mai179-Gal4 [27 (link)] was obtained from G. Korge (Berlin, Germany). This drives expression in a subset of neurons in the brain including some neurosecretory cells. The c929-Gal4 line was obtained from P. H. Taghert (St. Louis, MO). This Gal4 reveals the Drosophila dimmed (dimm) gene that encodes a bHLH protein, DIMM, known to be required for the differentiation of peptidergic neurons and endocrine cells [24 (link)]. Finally, UAS-cd8gfp flies (from Bloomington Stock center) were used as targets to express GFP.
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Publication 2008
Basic Helix-Loop-Helix Transcription Factors Brain Cells Choline O-Acetyltransferase Cold Temperature Diptera Drosophila Drosophila melanogaster Endocrine Cells Genes Genotype Glutamate Decarboxylase glutamate decarboxylase 1 (brain, 67kDa), human Immunocytochemistry In Situ Hybridization LacZ Genes Mushroom Bodies Neurons neuropeptide F Strains Tyrosine 3-Monooxygenase Tyrosine Decarboxylase Vesicular Glutamate Transport Proteins
The extraction of crude polysaccharide from the fruiting body of the mushroom D. indusiata was done following the procedure reported previously [55 (link)]. Briefly, the fruiting bodies of D. indusiata were dried in a hot air-drying oven at 45 °C and crushed into powder using a tissue triturator. The powder of fruiting body was extracted by using boiling water and the collected supernatant was then concentrated at 60 °C using a rotary evaporator. After that, the concentrated supernatant was deproteinated several times by the Sevag method [56 ] and the protein content was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) following the manufacturer’s instructions. The resulting solution was ethanol precipitated, dried using vacuum freeze drying and collected as a crude polysaccharide (DIP).
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Publication 2018
bicinchoninic acid Biological Assay Ethanol Human Body Mushroom Bodies Polysaccharides Powder Proteins Tissues Vacuum
Flies of the genotype RN2-Flp, Tub-FRT-CD2-FRT-Gal4, UAS mCD8-GFP were used in our experiments to label the CSDn. The RN2-Flp transgene was constructed by fusing the regulatory region of the evenskipped (eve) locus necessary for expression in RP2, aCC and pCC neurons [26 (link)] to the Flippase coding region. The construct on the third chromosome was allowed to undergo recombination with a strain carrying the P-(Tub-FRT-CD2-FRT-Gal4) [62 (link)] cassette and also a UAS-mCD8-GFP transgene [63 (link)]. In flies of the RN2-Flp, Tub-FRT-CD2-FRT-Gal4, UAS-mCD8-GFP genotype, expression of Flippase in specific cells induces recombination at the FRT sites, hence inducing expression of GFP under Tub-Gal4 control. The UAS-EcR-B1-d655W650A stock (referred to as UAS-EcR-B1W650A [31 (link),64 (link)]) was obtained from the Bloomington Drosophila Stock center, Indiana. UAS-TNT-G and UAS -IMPTNT-V stocks were kindly provided by Sean Sweeney [33 (link)]. The IMPTNT-V is mutated in LC2V233 to V237 mutation, rendering the toxin essentially ineffective [33 (link)]. Expression of the inactive toxin was used as a control in all experiments. The UAS-shits1/UAS-shits1; +/+;UAS-shits1/UAS-shits1stock [34 (link)], expresses a temperature sensitive form of Dynamin, under GAL4 control, at 29°C, which results in a block in endocytosis. The construction of the y, w; UAS-EGFP-Kir2.1 transformant has been described in [35 (link)]. Targeted expression leads to an inward flux of K+ ions that hyperpolarizes the resting membrane, resulting in silencing of neuronal activity. The lz3 stock was obtained from R Stocker [65 (link)]. Mushroom body marking was achieved using the mb247-DsRed stock kindly provided by Andre Fiala. UAS-nSybGFP was a gift from Mani Ramaswami, UAS-nod:lacZ from Clark et al. [23 (link)] and UAS-Dlg from Ulrich Thomas [25 (link)]. The UAS-Syt-GFP stock, Antennapaedia (Antp6), RN2-Gal4 and the temperature sensitive allele ddcts2 were obtained from the Bloomington Stock Centre, Indiana.
All fly stocks were grown on standard cornmeal medium at 25°C. White prepupae (0 hours APF) were collected and allowed to develop on moist filter paper. RN2-Flp, Tub-FRT-CD2-FRT Gal4, UAS mCD8-GFP pupae take about 100 hours to eclose when grown in our laboratory. All perturbations were done at 25°C except for the experiments with shits1 ectopic expression, for which animals were reared at 29°C.
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Publication 2007
Alleles Animals Cells Chromosomes Drosophila Dynamins Ectopic Gene Expression Endocytosis Genotype Ions LacZ Genes Mushroom Bodies Mutation Neurons Pupa Recombination, Genetic Regulatory Sequences, Nucleic Acid Strains Tissue, Membrane Toxins, Biological Transgenes

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Publication 2017
Adult Antibodies Brain Cloning Vectors Drosophila Human Body Immunoglobulins Immunohistochemistry Lens, Crystalline Microscopy, Confocal Mushroom Bodies Neurons Neuropil Vision

Most recents protocols related to «Mushroom Bodies»

Example 2

30 g of ethanol, 70 g of mushroom fruiting bodies and 10 g of fruit extract are added to 70 g of an aqueous solution of fermented mycelium to form a mixture. The mixture is heated to a temperature of approx. 100° C. The temperature of the mixture is maintained at 100° C. for approximately 7 days. The mixture is then cooled to room temperature and filtered to remove the remaining solid matter. The resulting solution is stored at approx. 4° C.

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Patent 2024
Ethanol Fruit Mushroom Bodies Mycelium Suby's G solution
The mushroom fruit body used in the experiments was dried by a hot-air dryer at 45 °C for 48 h and pulverized using a pulverizer. The mushroom extracts were prepared by using 80% methanol according to the method developed by Jang and Yoon [12 ]. 50 g of the fruit body power was immersed in 1 L of methanol (Ducksan Company, Korea) for 48 h to be extracted at room temperature twice. The extracts were filtered with filter paper (Advantec Toyo Co., No. 2., Japan), concentrated with a rotary evaporator, and freeze-dried (LP-10, Ilshin Bio Base Co., Korea) to obtain brown mushroom extract powder. The resulting mushroom extracts were kept at −70 °C to be used in the experiment.
Publication 2023
Agaricales Bio-Base Freezing Fruit Human Body Methanol Mushroom Bodies Powder
Determination of sample size: Brains were prepared for imaging in batches of 5–10. In initial batches, we assessed the variability of the manipulation, for example if we were trying to change Kenyon cell number, we looked at how variable the size of the Kenyon cell population was following the manipulation. We used this variability to determine how many batches to analyze so as to obtain enough informative samples. To avoid introducing statistical bias, we did not analyze the functional consequence of the manipulation until after completing all batches; for example, if the manipulation was intended to alter Kenyon cell claw numbers, we did not quantify odor responses until after completing all samples. Similarly, we did not measure the effect on other cell types (such as assessing projection neuron bouton phenotypes) until after completion of all samples. Genotypes or conditions being compared with one another were always prepared for staining together and imaged interspersed with one another to equalize batch effects except in occasional cases that have been highlighted in methods above. Since our developmental manipulations seemed to affect the mushroom body in each hemisphere independently and variably, we treated each hemisphere as an independent sample in our staining and functional imaging. In the case of hydroxyurea-treated animals, if one hemisphere was affected severely, the other one is likely affected to a similar extent but not necessarily equally, e.g. we observed KC clone counts of “[1,0]” or “[3,2]” but never “[4,0]”.
Criteria for exclusion, treatment of outliers: We excluded from analysis samples with overt physical damage to the cells or structures being measured. In figures and analyses, we treated outliers the same way as other data points. For in vivo functional imaging experiments, full criteria for inclusion and exclusion of each sample are discussed above under “Analysis of KC somatic odor responses” and “Analysis of MBON odor responses”.
Statistical tests applied are mentioned in each figure legend along with the p-value significance. To communicate our findings in the simplest and most complete way, we have displayed each data point for each sample to allow readers to assess effect size and significance directly. When sample size could be determined from the figures, we did not explicitly state it in the figure legends.
All statistical analysis were performed in GraphPad Prism, Excel, or R.
Publication Preprint 2023
Animals Brain Cells Claw Clone Cells Diploid Cell Genotype Hydroxyurea Mushroom Bodies Neurons Odors Phenotype Physical Examination prisma
To generate flies with reduced Kenyon cell numbers, we used an existing chemical method to ablate KC neuroblasts (de Belle and Heisenberg, 1994 (link); Elkahlah et al., 2020 (link); Sweeney et al., 2012 (link)). In Drosophila, 4 mushroom body neuroblasts from each hemisphere give rise to ~500 KCs each (Ito et al., 1997 (link)). Most neuroblasts pause their divisions during the first 8 hours after larval hatching (ALH), however MB neuroblasts continue to divide. Therefore, by feeding larvae a mitotic poison hydroxyurea (HU) during this time window, Kenyon cell neuroblasts can specifically be ablated (de Belle and Heisenberg, 1994 (link)). We can achieve a wide range of KC numbers by tweaking the concentration and duration of HU application (Elkahlah et al., 2020 (link)). This allowed us to generate flies with 0 to 4 KC neuroblasts, and the effect on each hemisphere was independent of the other resulting in a large range of KC numbers. However, as we described previously, the most informative brains, with intermediate numbers of KC neuroblasts remaining, are difficult to obtain—most ablation batches have a preponderance of unaffected or fully ablated mushroom bodies.
The protocol was adapted from (Elkahlah et al., 2020 (link); Sweeney et al., 2012 (link)). We set up large populations of flies in cages two days prior to ablation and placed a 35 or 60 mm grape juice agar plate (Lab Express, Ann Arbor, MI) in the cage with a dollop of goopy yeast. One day prior to the ablation, we replaced the grape juice/yeast plate with a new grape juice/yeast plate. On the morning of the ablation, we removed the plate from the cage and discarded the yeast puck and any hatched larvae on the agar. We then monitored the plate for up to four hours, until many larvae had hatched. Larvae were washed off the plate using a sucrose solution, and eggs were discarded. Larvae were then strained in coffee filters, and submerged in hydroxyurea (Sigma, H8627) in a yeast:AHL mixture, or sham mixture without HU. Ablation condition was 10 mg/mL HU, given for 1 hour. One batch experienced 15 mg/mL HU, given for 1 hour; this was done to gather more data points with a lower KC clone count. Larvae were then strained through coffee filters again, rinsed, and placed in a vial or bottle of B food (for MBON functional imaging and immunohistochemistry) or Janelia food supplemented with 0.2 mM all-trans-retinal (for behavior) until eclosion. We opened a new container of hydroxyurea each month as it degrades in contact with moisture, and we found its potency gradually declined. We achieved a U-shaped distribution of the HU effect, with many samples unaffected and many with all four KC neuroblasts lost. Ablated animals along with the control group (sham) were shipped in temperature-controlled conditions to Janelia Research Campus for behavior. A digital thermometer was kept in each shipment to record the lowest and highest temperature experienced during shipping. Batches that experienced ~15-27 Celsius were used for experiments. Animals were either shipped as larvae or late pupae, and not as adults in order to give enough time window for using the appropriate age of adult flies for behavior experiments.
Publication Preprint 2023
Adult Agar Animals Brain Cells Clone Cells Coffee Drosophila Eggs Fever Fingers Food Grapes Hydroxyurea Immunohistochemistry Larva Mushroom Bodies Poisons Population Group Pupa Retina Sucrose Thermometers Yeast, Dried
We analyzed males for immunohistochemistry in our manipulations. For functional imaging experiments, we used mixed-sex populations, and did not observe any correlation with sex (not shown). The Y-arena behavior used females due to the size of the arena not being optimal for males. Therefore, the flies dissected post-behavior and used for calyx area quantification in those animals and Brp staining (Figures 1D, 2H, 6F) were also females. Sex differences in the fly are well-documented, including in our own previous work, and anatomic and physiologic sex differences have not been observed in the mushroom body (Brovkina et al., 2021 (link); Clowney et al., 2015 (link)). Any brains that appeared damaged from dissections, or those with the mushroom body region obscured due to insufficient tracheal removal, were not included in the analysis.
Researchers performing quantification could not generally be blinded to experimental condition due to the overt changes in neuron numbers and brain structures induced by our manipulations. However, analysis was performed blind to the goals of the experiment when possible, and quantitation of features on the anterior and posterior sides of the brain were recorded independent of one another and merged after all quantifications were completed. Moreover, many of our analyses make use of variation within an experimental condition or genotype, providing an additional bulwark against observational bias.
Publication Preprint 2023
Agaricales Animals Brain Dissection Females Genotype Immunohistochemistry Kidney Calices Males Mushroom Bodies Neurons Parts, Body physiology Population Group Trachea Training Programs Visually Impaired Persons

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More about "Mushroom Bodies"

Mushroom bodies are fascinating neuroanatomical structures found in the brains of many arthropods, including insects and crustaceans.
These complex neuropil regions play a crucial role in learning, memory, and sensory integration, making them a captivating area of research.
Composed of densely packed intrinsic neurons called Kenyon cells, mushroom bodies receive input from various sensory modalities and integrate this information to generate complex behaviors.
The precise functions and underlying neuronal circuitry of these enigmatic brain regions are still not fully understood, leaving many unanswered questions.
Exploring the evolution and diversity of invertebrate cognition through the study of mushroom bodies can provide valuable insights.
Techniques such as Vectashield, β-actin immunostaining, and RIPA buffer lysis can be employed to investigate the neuroanatomy and biochemistry of these structures.
Whatman No. 1 filter paper and POL 016 may also be useful for sample preparation and analysis.
Fluorescent labeling with Alexa 546 conjugates and LabVIEW software can aid in visualizing and analyzing the complex neuronal networks within mushroom bodies.
Rabbit anti-GFP antibodies can be used to study the expression of specific proteins.
Additionally, the use of antibiotics like penicillin and streptomycin can help maintain cell cultures for further experimentation.
By leveraging these tools and techniques, researchers can delve deeper into the workings of mushroom bodies, ultimately expanding our understanding of the remarkable complexity and diversity of invertebrate brains.
PubCompare.ai can be a valuable resource in this endeavor, helping to optimize research protocols and locate the best available information from literature, pre-prints, and patents.