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Representative Results






Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

Published: September 14th, 2018



1Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, 2Department of Biological Sciences, Graduate School of Science, The University of Tokyo

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.

We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

The European honeybee (Apis mellifera L.) is a eusocial insect, and female bees show caste-dependent reproduction and age-dependent division of labor. For example, in the sterile caste of bees referred to as 'workers', younger individuals feed the broods while older ones forage nectar and pollen outside the hive1. Learning and memory ability is critically important in the life of the honeybee, because foragers must repeatedly go back and forth between food sources and their nest and then communicate the locations of good food sources to their nestmates through dance communication1. Previous stu....

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1. Capture of Foraging Honeybees

  1. Purchase honeybee colonies from a local distributor.
  2. Using an insect net, catch forager bees which return to the hive with pollen bags on their hind legs. Transfer the bees to a standard 50-mL plastic conical tube and cap the tube (Figure 1). Put the tube on ice to anesthetize the bees.
    NOTE: Wear the designated jackets for beekeeping to avoid bee stings. Nursing bees can also be collected depending on the experiment. To catch the.......

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Protein Concentrations in Brain Homogenates:
We prepared homogenates using forager bees. The calculated protein concentrations in the original homogenates are shown in Figure 3. The approximate protein concentrations in the original homogenate were as follows: 1.5 mg/mL in the MBs and 2.3 mg/mL in other brain regions. We used two bees per lot and six lots were analyzed.

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The biochemical examination of protein activity is profoundly important for understanding molecular signaling in the brain, because the activity of an enzyme is affected by various molecules, such as substrates and inhibitors, and can, thus, change along with animal behavior (e.g., learning and memory)5. In honeybee studies, enzymes such as cyclic AMP-dependent protein kinase A, cyclic GMP-dependent protein kinase, PKC, phosphorylated CaMKII, and adenylate cyclase are reported to be diffe.......

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Figure 4B - 4D was modified from Suenami et al.24 with the permission of Biology Open. The authors are grateful to the publisher for the permission. This work was supported by the Human Frontier Science Program (RGY0077/2016) to Shota Suenami and Ryo Miyazaki.


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Name Company Catalog Number Comments
Pierce BCA Protein Assay Kit ThermoFisher Scientific 23227 The reagent kit for measurement of protein concentration
Pierce Bovine Serum Albumin Standard Ampules 2mg/mL ThermoFisher Scientific 23209 The standard samples used in BCA assay
Paraffin wax GC 13B1X00155000141 Dental wax used as dissection stage
Insect pin Shiga No. 0 Stainless, solid head
PLCglow KXT Bio KCH-0001 A fluorogenic substrate of PLC
384-well microplate Corning 4511 Low-volume, round-bottom plate in black color
Gemini EM microplate reader Molecular Devices
Edelfosine Santa Cruz Biotechnology sc-201021 pan-PLC inhibitor
Neomycin sulfate Santa Cruz Biotechnology sc-3573 pan-PLC inhibitor

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