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.

1. Capture of Foraging Honeybees
<ol>
	<li>Purchase honeybee colonies from a local distributor.</li>
	<li>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...

<ol>
	<li>Winston, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biology of the Honey Bee. , (1991).</li><li>Szyszka, P., Galkin, A., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+and+non-associative+plasticity+in+Kenyon+cells+of+the+honeybee+mushroom+body.">Associative and non-associative plasticity in Kenyon cells of the honeybee mushroom body.</a> Frontiers in Systems Neuroscience. 2, 3 (2008).</li><li>M&#252;&#223;ig, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+disruption+of+the+NMDA+receptor+subunit+NR1+in+the+honeybee+brain+selectively+impairs+memory+formation.">Acute disruption of the NMDA receptor subunit NR1 in the honeybee brain selectively impairs memory formation.</a> The Journal of Neuroscience. 30 (23), 7817-7825 (2010).</li><li>Devaud, J. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+substrate+for+higher-order+learning+in+an+insect:+mushroom+bodies+are+necessary+for+configural+discriminations.">Neural substrate for higher-order learning in an insect: mushroom bodies are necessary for configural discriminations.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (43), E5854-E5862 (2015).</li><li>Gr&#252;nbaum, L., M&#252;ller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a+specific+olfactory+memory+leads+to+a+long-lasting+activation+of+protein+kinase+C+in+the+antennal+lobe+of+the+honeybee.">Induction of a specific olfactory memory leads to a long-lasting activation of protein kinase C in the antennal lobe of the honeybee.</a> The Journal of Neuroscience. 18 (11), 4384-4392 (1998).</li><li>Kamikouchi, A., Takeuchi, H., Sawata, M., Natori, S., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentrated+expression+of+Ca2+/calmodulin-dependent+protein+kinase+II+and+protein+kinase+C+in+the+mushroom+bodies+of+the+brain+of+the+honeybee+Apis+mellifera+L.">Concentrated expression of Ca2+/calmodulin-dependent protein kinase II and protein kinase C in the mushroom bodies of the brain of the honeybee Apis mellifera L.</a> The Journal of Comparative Neurology. 417 (4), 501-510 (2000).</li><li>Sen Sarma, M., Rodriguez-Zas, S. L., Hong, F., Zhong, S., Robinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+profiling+of+central+nervous+system+regions+in+three+species+of+honey+bee+during+dance+communication+behavior.">Transcriptomic profiling of central nervous system regions in three species of honey bee during dance communication behavior.</a> PLoS ONE. 4 (7), e6408 (2009).</li><li>Kaneko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+analysis+of+the+expression+of+futsch,+tau,+and+MESK2+homologues+in+the+brain+of+the+European+honeybee+(Apis+mellifera+L.).">In situ hybridization analysis of the expression of futsch, tau, and MESK2 homologues in the brain of the European honeybee (Apis mellifera L.).</a> PLoS ONE. 5 (2), e9213 (2010).</li><li>Kaneko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+middle-type+Kenyon+cells+in+the+honeybee+brain+revealed+by+area-preferential+gene+expression+analysis.">Novel middle-type Kenyon cells in the honeybee brain revealed by area-preferential gene expression analysis.</a> PLoS ONE. 8 (8), e71732 (2013).</li><li>Pasch, E., Muenz, T. S., R&#246;ssler, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+is+differentially+localized+in+synaptic+regions+of+kenyon+cells+within+the+mushroom+bodies+of+the+honeybee+brain.">CaMKII is differentially localized in synaptic regions of kenyon cells within the mushroom bodies of the honeybee brain.</a> The Journal of Comparative Neurology. 519 (18), 3700-3712 (2011).</li><li>Suenami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+differentiation+of+Kenyon+cell+subtypes+using+three+mushroom+body-preferential+genes+during+metamorphosis+in+the+honeybee+(Apis+mellifera+L.).">Analysis of the differentiation of Kenyon cell subtypes using three mushroom body-preferential genes during metamorphosis in the honeybee (Apis mellifera L.).</a> PLoS ONE. 11 (6), e0157841 (2016).</li><li>Farooqui, T., Robinson, K., Vaessin, H., Smith, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+early+olfactory+processing+by+an+octopaminergic+reinforcement+pathway+in+the+honeybee.">Modulation of early olfactory processing by an octopaminergic reinforcement pathway in the honeybee.</a> The Journal of Neuroscience. 23 (12), 5370-5380 (2003).</li><li>Matsumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+nucleotide-gated+channels,+calmodulin,+adenylyl+cyclase,+and+calcium/calmodulin-dependent+protein+kinase+II+are+required+for+late,+but+not+early,+long-term+memory+formation+in+the+honeybee.">Cyclic nucleotide-gated channels, calmodulin, adenylyl cyclase, and calcium/calmodulin-dependent protein kinase II are required for late, but not early, long-term memory formation in the honeybee.</a> Learning &amp; Memory. 21 (5), 272-286 (2014).</li><li>Scholl, C., K&#252;bert, N., Muenz, T. S., R&#246;ssler, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+knockdown+affects+both+early+and+late+phases+of+olfactory+long-term+memory+in+the+honeybee.">CaMKII knockdown affects both early and late phases of olfactory long-term memory in the honeybee.</a> Journal of Experimental Biology. 218, 3788-3796 (2015).</li><li>Miyata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficient+long-term+synaptic+depression+in+the+rostral+cerebellum+correlated+with+impaired+motor+learning+in+phospholipase+C+&#946;4+mutant+mice.">Deficient long-term synaptic depression in the rostral cerebellum correlated with impaired motor learning in phospholipase C &#946;4 mutant mice.</a> European Journal of Neuroscience. 13 (10), 1945-1954 (2001).</li><li>Koh, H. -. Y., Kim, D., Lee, J., Lee, S., Shin, H. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+social+behavior+and+sensorimotor+gating+in+mice+lacking+phospholipase+C&#946;1.">Deficits in social behavior and sensorimotor gating in mice lacking phospholipase C&#946;1.</a> Genes, Brain and Behavior. 7 (1), 120-128 (2008).</li><li>Quan, W. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+behaviors+and+prepulse+inhibition+in+phospholipase+C&#949;-/-+mice.">Characteristics of behaviors and prepulse inhibition in phospholipase C&#949;-/- mice.</a> Neurology,Psychiatry and Brain Research. 18 (4), 169-174 (2012).</li><li>Rioult-Pedotti, M. -. S., Pekanovic, A., Atiemo, C. O., Marshall, J., Luft, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+promotes+motor+cortex+plasticity+and+motor+skill+learning+via+PLC+activation.">Dopamine promotes motor cortex plasticity and motor skill learning via PLC activation.</a> PLoS ONE. 10 (5), e0124986 (2015).</li><li>Ghosh, A., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+neurons:+molecular+mechanisms+and+cellular+consequences.">Calcium signaling in neurons: molecular mechanisms and cellular consequences.</a> Science. 268 (5208), 239-247 (1995).</li><li>Smrcka, A. V., Brown, J. H., Holz, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phospholipase+C&#949;+in+physiological+phosphoinositide+signaling+networks.">Role of phospholipase C&#949; in physiological phosphoinositide signaling networks.</a> Cellular Signalling. 24 (6), 1333-1343 (2012).</li><li>Dusaban, S. S., Brown, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLC&#949;+mediated+sustained+signaling+pathways.">PLC&#949; mediated sustained signaling pathways.</a> Advances in Biological Regulation. 57, 17-23 (2015).</li><li>Elgersma, Y., Sweatt, J. D., Giese, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+genetic+approaches+to+investigating+calcium/calmodulin-dependent+protein+kinase+II+function+in+plasticity+and+cognition.">Mouse genetic approaches to investigating calcium/calmodulin-dependent protein kinase II function in plasticity and cognition.</a> The Journal of Neuroscience. 24 (39), 8410-8415 (2004).</li><li>Giese, K. P., Mizuno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+protein+kinases+in+learning+and+memory.">The roles of protein kinases in learning and memory.</a> Learning &amp; Memory. 20 (10), 540-552 (2013).</li><li>Suenami, S., Iino, S., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+inhibition+of+phospholipase+C+in+the+brain+attenuates+early+memory+formation+in+the+honeybee+(Apis+mellifera+L.).">Pharmacologic inhibition of phospholipase C in the brain attenuates early memory formation in the honeybee (Apis mellifera L.).</a> Biology Open. 7 (1), (2018).</li><li>Zhu, L., McKay, R. R., Shortridge, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+expression+of+phospholipase+C+encoded+by+the+norpA+gene+of+Drosophila+melanogaster.">Tissue-specific expression of phospholipase C encoded by the norpA gene of Drosophila melanogaster.</a> The Journal of Biological Chemistry. 268 (21), 15994-16001 (1993).</li><li>Huang, W., Hicks, S. N., Sondek, J., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorogenic,+small+molecule+reporter+for+mammalian+phospholipase+C+isozymes.">A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes.</a> ACS Chemical Biology. 6 (3), 223-228 (2011).</li><li>Yoshioka, T., Inoue, H., Hotta, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+phosphatidylinositol+phosphodiesterase+in+the+head+of+a+Drosophila+visual+mutant,+norpA+(no+receptor+potential+A).">Absence of phosphatidylinositol phosphodiesterase in the head of a Drosophila visual mutant, norpA (no receptor potential A).</a> The Journal of Biochemistry. 97 (4), 1251-1254 (1985).</li><li>Janjanam, J., Chandaka, G. K., Kotla, S., Rao, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLC&#946;3+mediates+cortactin+interaction+with+WAVE2+in+MCP1-induced+actin+polymerization+and+cell+migration.">PLC&#946;3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration.</a> Molecular Biology of the Cell. 26 (25), 4589-4606 (2015).</li><li>Fiala, A., M&#252;ller, U., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+downregulation+of+protein+kinase+A+during+olfactory+learning+using+antisense+technique+impairs+long-term+memory+formation+in+the+honeybee,+Apis+mellifera.">Reversible downregulation of protein kinase A during olfactory learning using antisense technique impairs long-term memory formation in the honeybee, Apis mellifera.</a> The Journal of Neuroscience. 19 (22), 10125-10134 (1999).</li><li>Thamm, M., Scheiner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKG+in+honey+bees:+spatial+expression,+Amfor+gene+expression,+sucrose+responsiveness,+and+division+of+labor.">PKG in honey bees: spatial expression, Amfor gene expression, sucrose responsiveness, and division of labor.</a> The Journal of Comparative Neurology. 522 (8), 1786-1799 (2014).</li><li>Balfanz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+transmembrane+adenylyl+cyclases+from+the+honeybee+brain.">Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.</a> Insect Biochemistry and Molecular Biology. 42 (6), 435-445 (2012).</li><li>Lopez, I., Mak, E. 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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...

The European honeybee (Apis mellifera L.) is a eusocial insect, and female bees show caste-dependent reproduction and&#160;age-dependent division of labor. For example, in the sterile caste of bees referred to as &#39;workers&#39;, younger individuals feed the broods while older ones&#160;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 PLC&#946;, PLC&#947;, and PLC&#949;, 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 PLC&#946; and PLC&#949; 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:479px;" width="478">
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:113px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:101px;">ThermoFisher Scientific</td>
			<td style="width:139px;">23227</td>
			<td style="width:125px;">The reagent kit for measurement of protein concentration</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:113px;">Pierce Bovine Serum Albumin Standard Ampules 2mg/mL</td>
			<td style="width:101px;">ThermoFisher Scientific</td>
			<td>23209</td>
			<td style="width:125px;">The standard samples used in BCA assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Paraffin wax</td>
			<td style="width:101px;">GC</td>
			<td>13B1X00155000141</td>
			<td style="width:125px;">Dental wax used as dissection stage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Insect pin</td>
			<td style="width:101px;">Shiga</td>
			<td>No. 0</td>
			<td style="width:125px;">Stainless, solid head</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">PLCglow</td>
			<td style="width:101px;">KXT Bio</td>
			<td>KCH-0001</td>
			<td style="width:125px;">A fluorogenic substrate of PLC</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:113px;">384-well microplate</td>
			<td style="width:101px;">Corning</td>
			<td>4511</td>
			<td style="width:125px;">Low-volume, round-bottom plate in black color</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:113px;">Gemini EM microplate reader</td>
			<td style="width:101px;">Molecular Devices</td>
			<td></td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Edelfosine</td>
			<td style="width:101px;">Santa Cruz Biotechnology</td>
			<td>sc-201021</td>
			<td style="width:125px;">pan-PLC inhibitor</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:113px;">Neomycin sulfate</td>
			<td style="width:101px;">Santa Cruz Biotechnology</td>
			<td>sc-3573</td>
			<td style="width:125px;">pan-PLC inhibitor</td>
		</tr>
	</tbody>
</table>

detection of phospholipase c activity in the brain homogenate from the honeybee

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.

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.
<stron...

Watch this Scientific Journal Video about Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee at JoVE.com

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

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 ...

This method can help answer key questions in the neuroscience field such as whether and how PLC activities in different brain regions are involved in brain function of the honeybee.The main advantage of this technique is that researchers can easily preform an experiment with the standard laboratory facilities as this protocol requires only basic manipulations.To begin, place a 50ml tube containing honeybees on ice for 30 minutes to anesthetize the bees.Next, prepare the dissection stage by folding a piece of dental wax in half and placing it in a plastic dish.Under a binocular microscope, use tweezers to separate the bee's head from its body.Then, fix the head on the dental wax and insert insect pins at the base of each antenna to hold the interior of the head in an upward position.Next, pour enough saline solution over the head to cover it.Using tweezers remove the antenna.After this, use a scalpel to make a horizontal cut near the bases of the antenna and a longitudinal cut at the border of the compound eyes.Make a horizontal cut at the top of the head and remove the cuticle to open a window over the brain.Next, use tweezers to remove the retin eye from the ossicle and the trache from the interior surface of the brain.Then, insert the tweezers directly under the eye cuticle to remove the connective tissue between the cuticle of the compound eyes and retin eye.After that, remove the cuticle and retin eye of the compound eyes.Next, use the tweezers to carefully remove the remaining trache from the interior surface of the brain.Then, cut the connective tissue between the brain and around the tissues and dissect the brain from the head capsule.Using the tweezers, peel the trache off of the posterior surface of the brain.Then use a scalpel to cut the connection between the mushroom bodies or MBs and other brain regions next to the vertical lobes.Place the dissected MBs and any remaining brain tissues into 1.5ml tubes and freeze them rapidly with dry ice.First, add 10ml of homogenization buffer to the frozen brain tissue.Use a plastic peselle to manually homogenize the tissue in the 1.5ml tube.After this, transfer the homogenized tissue solution to the next tissue in the lot and repeat the homogenization process.Then, add 30

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Research

JoVE Journal

Biochemistry

7.7K visualizações.  National Institute of Advanced Industrial Science and Technology. Este método pode ajudar a responder a perguntas-chave no campo da neurociência, como se e como as atividades de PLC em diferentes regiões cerebrais estão envolvidas na função cerebral da abelha. A principal vantagem dessa técnica é que os pesquisadores podem facilmente pré-formar um experimento com as instalações de laboratório padrão, pois este protocolo requer apenas manipulações básicas. Para começar, coloque um tubo de 50ml contendo abelhas no gelo por 30 minutos para ane...