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 studies demonstrated that the MB, a higher brain center in insects, is involved in the learning and memory ability of the honeybee2,3,4. Differentially expressed genes and proteins have been identified in various brain regions of the honeybee5,6,7,8,9,10,11, suggesting that they are related to the unique functions of each brain region. Although the pharmacologic inhibition or activation of a protein of interest is a well-used approach to reveal the function of the protein in honeybee behavior12,13,14, it is unknown whether all drugs have functional effects in different regions of the honeybee brain. The validation of the functions of such drugs will strengthen conclusions in studies of behavioral pharmacology.
Here, we focus on PLC, one of the enzymes implicated in mouse cognition15,16,17,18. PLC triggers calcium signaling by degrading phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)19,20,21. IP3 opens IP3 receptors on the endoplasmic reticulum (ER), leading to the release of calcium ions from the ER. The released calcium activates both calcium/calmodulin-dependent protein kinase II (CaMKII) with calmodulin and protein kinase C (PKC) in the presence of DAG. Both protein kinases are involved in learning and memory22,23, consistent with the involvement of PLC in this process. PLCs are categorized into subtypes, including PLCβ, PLCγ, and PLCε, based on their structures20. Each PLC subtype is activated in a different context20, and genes encoding those subtypes are differentially expressed in different tissues. We previously demonstrated that honeybee MBs express genes encoding PLCβ and PLCε subtypes at higher levels than the remaining brain regions24, and that two pan-PLC inhibitors (edelfosine and neomycin sulfate [neomycin]) decrease PLC activity in different brain regions and, indeed, affect the learning and memory ability of the honeybee24.
Traditionally, the enzymatic activity of PLC has been measured using radiolabeled PIP225, which requires appropriate training, equipment, and facilities. Recently, a synthetic fluorogenic substrate of PLC has been established26, making it easy to assess PLC activity in the standard laboratory. Here, we present a detailed protocol to detect PLC activity in different brain regions of the honeybee using the fluorogenic substrate and to subsequently test the inhibitory effects of edelfosine and neomycin on PLC in these tissues. Because the protocol requires only basic manipulations, it may be applicable to studies of PLC activity in other tissues or brain areas in bees allocated to different social tasks.
1. Capture of Foraging Honeybees
2. Dissection of the Honeybee Brain
3. Preparation of Brain Homogenates
4. PLC Reaction in Brain Homogenates
5. Detection of Fluorescence Resulting from PLC Activity
6. Test of the Inhibitory Action of Pharmacologic Agents
7. Statistical Analysis
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.
Detection of PLC Activity in the Brain Homogenates:
In the pilot experiment, we detected higher fluorescence in other brain regions than in the MBs. Therefore, we repeated the preliminary experiments using the homogenate of the other brain regions and determined the reaction time (i.e., 30 min) and protein amount (i.e., 1.3 µg). The result of the reaction under these conditions is shown in Figure 4A. The reaction mixtures containing both tissue homogenate and the fluorogenic substrate exhibited > 4.2-fold higher fluorescence than the control mixtures containing either the tissue homogenate or the fluorogenic substrate, suggesting that PLC activity was detected in the reaction mixtures. Relative fluorescence was approximately 3.4-fold higher in other brain regions than in the MBs (Figure 4B).
Analysis of the Inhibitory Effects of Pharmacologic Agents on PLC Activity:
To examine the effects of the pan-PLC inhibitors edelfosine and neomycin on PLC activity, we performed the reaction in the presence of 1.0 mmol/L edelfosine or 0.55 mmol/L neomycin, using the same homogenate as analyzed above. In the presence of edelfosine, the fluorescence level decreased to approximately 6.0% and 5.4% in the MBs and other brain regions, respectively, compared to controls without edelfosine (Figure 4C). Neomycin treatment reduced the fluorescence level to 44% and 20% that of untreated controls in the MBs and other brain regions, respectively (Figure 4D).
Figure 1: Capturing a forager honeybee. A forager returning to her hive was captured in an insect net, and she was confined into a 50-mL plastic conical tube. Please click here to view a larger version of this figure.
Figure 2: Schematic representation of the dissection procedure. (A) Axes of the honeybee brain mentioned in the protocol are shown. The bee from the head to the anterior thorax is viewed from the lateral side. (B – K) Photos and illustrations of the dissection procedures are shown. See the main text for details. Only the head of the honeybee is presented. An illustration of the tracheae is omitted. MBs = mushroom bodies. The scale bars correspond to 1 mm. Please click here to view a larger version of this figure.
Figure 3: Protein concentrations in brain tissue homogenates. Protein concentrations in the original homogenates were measured by BCA assay and calculated. The mean concentrations with standard deviations are shown. Two forager bees were used for each lot, and six lots were analyzed. MBs = mushroom bodies. Please click here to view a larger version of this figure.
Figure 4: Fluorescence detected in reactions. (A) This panel shows fluorescence in each tissue with or without the fluorogenic substrate. The reaction was performed for 30 min using 1.3 µg of protein. Fluorescence was measured by the microplate reader. The values of the sample wells were corrected by empty wells and shown in arbitrary units (AUs). (B) This panel shows a comparison of fluorescence between brain tissues. The data in panel A were corrected for control mixtures by subtraction and normalized by the calculated fluorescence in the MBs. * P < 0.005, Mann-Whitney's U test. Panels C and D show relative fluorescence in the presence of (C) 1.0 mmol/L edelfosine and (D) 0.55 mmol/L neomycin. The same homogenates used in panels A and B were analyzed. The fluorescence values were normalized by the results of the control experiment without drug treatment for each tissue. The data of the control reactions in panel C are the same as in panel B. In panel D, all homogenates were analyzed again in a different experiment. The mean fluorescence values with standard deviations are shown. Two bees were used for each lot, and six lots were analyzed. * P < 0.05, Wilcoxon signed-ranks test. No Hg = control mixture not containing homogenate; MBs = mushroom bodies. Panels B – D were modified from Suenami et al.24 with the publisher's permission. Please click here to view a larger version of this figure.
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 differentially expressed in various brain regions based on immunohistochemistry5,10,29,30,31. Differences in enzymatic activity among brain regions, however, are only partially reported6. Here, we described a detailed protocol for detecting PLC activity and assessing the inhibitory effects of pharmacologic agents in the MBs and other brain regions.
There are some possible factors interfering with fluorescence detection. First, it is important to protect the protein from degradation when performing biochemical assays. In the protocol described here, a quick dissection and freezing of the brain are required. It is recommended that tissue samples are frozen and stored soon after each cycle of dissection. A minimization of the thaw/freeze cycle of the homogenate is also crucial to prevent the deterioration of the protein.
In addition to protein degradation, background fluorescence and absorbance in the reaction mixture may affect the results in this assay system, because PLC activity is detected by a fluorescence signal. For example, neomycin dissolved in water has a yellow color that affects fluorescence detection. Although we used 0.55 mmol/L neomycin, a 1000-fold dilution of the stock solution, a further optimization of the concentration might be required. Moreover, contamination by other tissues may also influence the result. The retina, which detects visual stimuli and contains pigments, comprises one tissue type that potentially interferes with the assay.
With the present protocol, we detected higher PLC activity in other brain regions than in the MBs24. This was inconsistent with the result of quantitative reverse-transcription PCR analysis, which revealed that the MBs express higher levels of PLC genes than other brain tissues24. This contradiction might be due to WH-15, which is a free-floating substrate26, and the fact that we did not distinguish the membrane and cytosolic fractions of the brain homogenates. Considering that PLCβ and PLCε are membrane-associated enzymes32 and can interact with the endogenous PIP2 substrate, the amount of the membrane fraction in the homogenate likely affects the reaction between PLC and WH-15. Another possible explanation is that the PLC concentration might be higher in the other brain regions than in the MBs due to differences in protein production and/or degradation rates. Hence, bona fide PLC activity in the brain must be clarified further by additional experiments, such as by using a recently reported new substrate incorporated into the membrane33, analyzing the activity of membrane-associated and cytosolic PLCs separately, or quantifying the content of PLCs or PIP2 in each brain tissue.
Taking the above points into account, the PLC assay system described here can be expanded to measure PLC activity in different tissues not assessed here, such as the digestive tract, muscle, and reproductive organ. It is also possible to compare PLC activity between the brains of forager and nurse bees to evaluate the involvement of PLC activity in different social roles.
Overall, the assay system presented here is a feasible option for detecting PLC activity in tissue homogenates, because it can be performed with standard laboratory equipment, while a traditional approach using radiolabeled PIP2 requires specialized facilities, training, and equipment for radioisotopes. Taking advantage of the system with further modifications will deepen our understanding of the molecular mechanisms underlying the honeybee's complex behavior.
The authors have nothing to disclose.
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.
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 |