This work was supported by a grant from the German Federal Ministry of Education and Research (BMBF) within the Bernstein Focus: Neuronal Basis of Learning (BFNL) to D.E. We thank Randolf Menzel for valuable comments on an earlier version of the manuscript.

1. Catching Bees from the Hive
<ol>
<li>One day before the experiment starts, between 2 and 4 p.m., bees leaving the hive are caught. To do so, a UV light-permeable plexiglass pyramid (height = 30 cm, apex 3,5 x 3, 5 cm, base 18 x 18 cm), which is closable at the apex and the base, is held at a 20-30 cm distance in front of the hive entrance with the base open and the apex closed so that bees leaving the hive enter the base of the pyramid. The base is then closed and the captured bees are brought into the lab for further handling.</li>
</ol>
2. 	Transferring Bees from the Pyramid Into Glass Vials
<ol>
<li>In the lab, the pyramid is placed on its base. The walls of the pyramid are darkened (e.g. with a towel) but the apex is left uncovered. Because of their positive phototaxis, bees will leave the pyramid through the apex when opened. One by one, bees are transferred from the pyramid into glass vials by holding the vials over the open apex. One vial is used per bee. Therefore, the apex is closed when one bee enters the vial.</li>
</ol>
3. 	Harnessing Bees in Tubes
<ol>
<li>Bees are immobilized by cooling them in the glass vials on ice for 2.5-3.5 min. It is advisable to watch the bee and remove it from the ice as soon as it stops moving.</li>
<li>A single immobilized bee is harnessed in a small plastic tube with sticky tape, such that it is able to move its proboscis freely but not its head, thorax or legs. It is important that the neck is not compressed.</li>
<li>Every bee fixed in a plastic tube is put into a numbered borehole on a rack for better handling and identification. After it has been removed for conditioning or memory retrieval the tube is always returned to the exact same borehole.</li>
</ol>
4. 	Feeding Bees
<ol>
<li>On the first evening (4-6 p.m.), after being caught, bees are fed to satiation with sucrose solution (0,88 M, white refined household sugar dissolved in tap water). To feed a bee, its PER is elicited by touching its antennae with sucrose solution and the animal is allowed to consume one droplet (4 &mu;L) of sucrose solution. Bees are fed one after another repeatedly until they no longer show a fast and reliable PER when their antennae are touched with sucrose solution.</li>
<li>On each subsequent evening (4-6 p.m.) of the experiment bees are fed one after another four times with 1 droplet of sucrose solution (4 &mu;L, 0,88 M, white refined sugar solved in tap water). It is important that sucrose solution is not smeared on the bee's antennae or the proboscis during feeding and that the tubes are not contaminated with sucrose solution. Bees should not be fed at or near the conditioning site to exclude the possibility of associating the training context with the sucrose stimulus.</li>
</ol>
5. 	Keeping Harnessed Bees Overnight
<ol>
<li>Bees are kept overnight in a plastic bowl at room temperature. Tap water is filled into the bowl (approximately 0,5-1 cm high). Racks with the harnessed bees are placed in the bowl above the waterline using ELISA plates as a platform. Finally, the bowl is covered with cardboard.</li>
</ol>
6. 	Olfactory Conditioning
On the first morning (10 a.m.) of the experiment olfactory conditioning is carried out. The conditioned stimulus (CS) is an odor, the unconditioned stimulus (US) is sucrose solution.
<ol>
<li>4 &mu;L of the odor (e.g. clove oil) are pipetted onto a round filter paper (1,3 cm diameter) which is then inserted into a 20 mL syringe. The odor is pipetted under a hood and filter tips are used to prevent contamination of the pipette.</li>
<li>The rack with harnessed bees is placed near the conditioning site 30 min before the conditioning procedure starts, but at a distance to the place in front of an exhaust pipe where single bees are conditioned.</li>
<li>Conditioning consists of three pairings of odor (the conditioned stimulus, CS) and sucrose solution (the unconditioned stimulus, US, 1,25 M) with an inter-trial interval (ITI) of 10 min. An acoustic signal delivered to the experimenter via an audio player ensures the precise timing of stimulus onset, stimulus offset, stimulus duration and placing. An acquisition trial starts with a 10 sec placement of the animal in front of an exhaust. Shortly before the end of the 10 sec the syringe containing the odor is placed 3 cm in front of the bee targeted at the bee's antennae. Subsequently, the odor is presented for 5 sec, by pushing 20 mL air through the syringe. After the first 3 sec the distal flagella of both antennae are touched with a sucrose solution-moistened toothpick and animals are allowed to lick the moistend toothpick for 4 sec. 13 sec after the CS stimulation ends, bees are taken out of the training context and placed back in the rack. Altogether one training trial lasts 28 sec. 
It is important that neither the bee's antennae nor the proboscis are covered with sucrose after conditioning. Therefore it has to be ensured that the toothpick is only moistened with sucrose solution and that no drops of sucrose solution form on the toothpick.</li>
<li>After conditioning the racks are placed back in the bowl.</li>
<li>The animals' behavior during the experiment, i.e. the occurence of the PER during placement and CS and US presentation, is monitored and noted down by the experimenter.</li>
</ol>
7. 	Memory Retention
<ol>
<li>Retention tests can be performed at any interval (min to days). The rack with bees is placed near the conditioning site 30 min before the memory test starts.</li>
<li>A memory test consists of 5 sec CS presentation without US presentation. The test starts with a 10 sec placement of the animal in front of the exhaust. Subsequently, the odor is presented for 5 sec as described above. The behavior of the animal, i.e. the occurence of the PER during placement and the CS presentation is noted down during the experiment.</li>
<li>At the end of the experiment the proboscis extention response is again elicited by touching the antennae with sucrose solution to ensure that the animal is still able to extend its proboscis.</li>
</ol>
8. Systemic Injection
The time point of systemic injection depends on the experimental design.
<ol>
<li>A small hole is made in the cuticula of the posterior part of the scutum next to the scutal fissure above the flight muscle17 by using a disposable hypodermic needle (21 G).</li>
<li>Using a glass capillary tube, 1 &mu;L solution is injected through the hole in the cuticula into the flight muscle. Trained experimenters are able to inject one bee every 30 sec, allowing a precise timing of injection and conditioning.</li>
</ol>
9. Feeding Bees During the Experiment
<ol>
<li>On the evening (4-6 p.m.) after conditioning, bees are fed one after another four times with 1 droplet of sucrose solution (4 &mu;L, 0,88 M, white refined sugar dissolved in tap water). It is important that sucrose solution is not smeared on the bee's antennae or the proboscis during feeding and that the tubes are not contaminated with sucrose solution. Bees should not be fed at or near the conditioning site to exclude the possibility of associating the training context with the sucrose stimulus.</li>
</ol>
10.	Data Collection and Data Analysis
<ol>
<li>Occurrence of the proboscis extension response during the experiment is monitored. A bee is scored positive if it extends its proboscis between the onset of the CS and the presentation of the US (during training) or during the CS presentation (during test phase), crossing a virtual line between the open mandibles.</li>
<li>To be included in the analysis animals must fulfill two criteria: they must survive the entire experiment and they must show the unconditioned proboscis extension response (PER) to sucrose solution at the end of the experiment. The percentage of bees showing the PER during acquisition and retention tests is plotted for each CS presentation.</li>
</ol>
11. Representative Results:
Two experiments are presented here.
In the first experiment we look at the impact of systemic injection of phosphate-buffered saline (PBS; in mM: 137 NaCl, 2,7 KCl, 10,1 Na2HPO4, 1,8 KH2PO4, pH 7,2) on learning and long-term memory formation. Three groups of bees were trained with three CS-US pairings with an inter-trial interval of 10 min: a non-treated group, a group that was injected with 1 &mu;L PBS 30 min before training, and a sham-injected group treated the same way as the PBS-injected group but without injecting PBS. 24 h after training memory was tested with one CS presentation in all three groups. The percentage of animals showing the conditioned response during conditioning increases significantly across the three training trials (Fig.1, ANOVA for repeated measurement for the factor time F2,378=340,456, p &lt; 0,05). No difference in PER could be observed between the three groups during conditioning (ANOVA for repeated measurement for the factor groups F2, 189 = 1,299, p &gt; 0,05). This holds true for a 24 h retention test (F2, 187 = 0,752, p&gt;0,05) when comparing non-treated animals with PBS-injected and sham-injected animals.
In the second experiment we demonstrate the inhibition of memory retention three and four days after training when the protein synthesis inhibitor anisomycin (10 mM) was systemically injected 30 min after three CS-US pairings. Comparison of an anisomycin-injected group with an PBS-injected group reveals a significant difference between groups during the retention test (Fig.2, ANOVA for repeated measurement for the factor group F1, 94=8,86, p &lt; 0,05). A major difference can be observed at the memory test on day 3 and day 4. This resembles the results of W&uuml;stenberg et al.18, who also conditioned bees with three CS-US pairings, albeit with a different protocol.
<img src="/files/ftp_upload/2282/2282fig1.jpg" alt="Figure 1" /> 
Figure 1. Cuticula lesion or injection of PBS does not alter aquisition and memory retention. Animals were trained with three CS-US pairings (acquisition). After 24 h, memory was tested (Memory Retention). Thirty minutes prior to training, a hole was made in the cuticula of two groups (lesion, PBS) and one of these groups was injected with 1 &mu;L PBS (PBS). A third group was left intact (no treatment). Presented is the percentage of animals for each group responding to odor presentation with a PER. An ANOVA does not reveal any significant differences between groups in the acquisition phase (F2, 189 = 1.299, p &gt; 0.05) or in memory retention (F2, 187 = 0.752, p&gt;0.05).
<img src="/files/ftp_upload/2282/2282fig2.jpg" alt="Figure 2" /> 
Figure 2. Injection of anisomycin 40 min after the last CS-US pairing reduces memory retention three days and four days after training. Animals were trained with three CS-US pairings (acquisition) and injected with 1 &mu;L 10 mM anisomycin (Aniso) or 1 &mu;L PBS 1 h after the first training trial. Memory was tested (Memory Retention) 24 h, 48 h, 72 h and 96 h later. Presented is the percentage of animals per group responding to odor presentation with a PER. The injection of anisomycin leads to a significant reduction of memory retention 72 h and 96 h after training (F1, 94=8.86, p &lt; 0.05) as revealed by an ANOVA.

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	<li>Menzel, R., Giurfa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+architecture+of+a+mini-brain:+the+honeybee.">Cognitive architecture of a mini-brain: the honeybee.</a> Trends Cogn Sci. 5, 62-71 (2001).</li><li>Menzel, R., Leboulle, G., Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+brains,+bright+minds.">Small brains, bright minds.</a> Cell. 124, 237-239 (2006).</li><li>Menzel, R., Kesner, R. P., Olton, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurobiology of comparative cognition. , (1990).</li><li>Bitterman, M. E., Menzel, R., Fietz, A., Schafer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical-Conditioning+of+Proboscis+Extension+in+Honeybees+(Apis-Mellifera).">Classical-Conditioning of Proboscis Extension in Honeybees (Apis-Mellifera).</a> J. Comp. Psyc. 97, 107-119 (1983).</li><li>Menzel, R., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+in+honeybees:+from+behavior+to+neural+substrates.">Learning and memory in honeybees: from behavior to neural substrates.</a> Annu. Rev. Neurosci. 19, 379-404 (1996).</li><li>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=Memory+dynamics+in+the+honeybee.">Memory dynamics in the honeybee.</a> Journal of Comparative Physiology A-Neuroethology Sensory Neural and Behavioral Physiology. 185, 323-340 (1999).</li><li>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=Searching+for+the+memory+trace+in+a+mini-brain,+the+honeybee.">Searching for the memory trace in a mini-brain, the honeybee.</a> Learn. Mem. 8, 53-62 (2001).</li><li>Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+formation+in+the+honeybee+(Apis+mellifera)+and+its+dependency+on+the+cAMP-protein+kinase+A+pathway.">Learning and memory formation in the honeybee (Apis mellifera) and its dependency on the cAMP-protein kinase A pathway.</a> Animal Biology. 56, 259-278 (2006).</li><li>Hourcade, B., Muenz, T. S., Sandoz, J. C., Rossler, W., Devaud, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+memory+leads+to+synaptic+reorganization+in+the+mushroom+bodies:+a+memory+trace+in+the+insect+brain.">Long-term memory leads to synaptic reorganization in the mushroom bodies: a memory trace in the insect brain.</a> J. Neurosci. 30, 6461-6465 (2010).</li><li>Denker, M., Finke, R., Schaupp, F., Grun, S., 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=Neural+correlates+of+odor+learning+in+the+honeybee+antennal+lobe.">Neural correlates of odor learning in the honeybee antennal lobe.</a> Eur. J. Neurosci. 31, 119-133 (2010).</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> Front Syst. Neurosci. 2, 3-3 (2008).</li><li>Okada, R., Rybak, J., Manz, G., 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=Learning-related+plasticity+in+PE1+and+other+mushroom+body-extrinsic+neurons+in+the+honeybee+brain.">Learning-related plasticity in PE1 and other mushroom body-extrinsic neurons in the honeybee brain.</a> J. Neurosci. 27, 11736-11747 (2007).</li><li>Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+activation+of+cAMP-dependent+protein+kinase+during+conditioning+induces+long-term+memory+in+honeybees.">Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees.</a> Neuron. 27, 159-168 (2000).</li><li>Locatelli, F., Bundrock, G., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+and+temporal+release+of+glutamate+in+the+mushroom+bodies+improves+olfactory+memory+in+Apis+mellifera.">Focal and temporal release of glutamate in the mushroom bodies improves olfactory memory in Apis mellifera.</a> J. Neurosci. 25, 11614-11618 (2005).</li><li>Stollhoff, N., Menzel, R., Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+recovery+from+extinction+depends+on+the+reconsolidation+of+the+acquisition+memory+in+an+appetitive+learning+paradigm+in+the+honeybee+(Apis+mellifera.">Spontaneous recovery from extinction depends on the reconsolidation of the acquisition memory in an appetitive learning paradigm in the honeybee (Apis mellifera.</a> J. Neurosci. 25, 4485-4492 (2005).</li><li>Stollhoff, N., Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consolidation+of+an+extinction+memory+depends+on+the+unconditioned+stimulus+magnitude+previously+experienced+during+training.">Consolidation of an extinction memory depends on the unconditioned stimulus magnitude previously experienced during training.</a> J. Neurosci. 29, 9644-9650 (2009).</li><li>Snodgrass, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy of the honeybee. , (1956).</li><li>Wustenberg, D., Gerber, B., 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=Short+communication:+long-+but+not+medium-term+retention+of+olfactory+memories+in+honeybees+is+impaired+by+actinomycin+D+and+anisomycin.">Short communication: long- but not medium-term retention of olfactory memories in honeybees is impaired by actinomycin D and anisomycin.</a> Eur. J. Neurosci. 10, 2742-2745 (1998).</li><li>Vollbehr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Orientierung+junger+Honigbienen+bei+ihrem+1.">Zur Orientierung junger Honigbienen bei ihrem 1.</a> Orientierungsflug. Zool. Jb. Physiol. 79, 33-69 (1975).</li><li>Scheiner, R., Erber, J., Page, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+learning+and+the+individual+evaluation+of+the+reward+in+honey+bees+(Apis+mellifera+L.).">Tactile learning and the individual evaluation of the reward in honey bees (Apis mellifera L.).</a> J. Comp Physiol [A]. 185, 1-10 (1999).</li><li>Sanchez, B. r. i. t. o., G, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+studies+on+tarsal+gustation+in+honeybees:+sucrose+responsiveness+and+sucrose-mediated+olfactory+conditioning.">Behavioral studies on tarsal gustation in honeybees: sucrose responsiveness and sucrose-mediated olfactory conditioning.</a> J. Comp Physiol A Neuroethol. Sens. Neural Behav. 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No conflicts of interest declared.

<ol>
<li>Catching bees: To collect experienced foraging bees, bees are caught either in the morning or in the afternoon, avoiding the time when young bees are on orientation flights19. Foragers collect either pollen or nectar. Differences between pollen and nectar foragers have been observed according to their sugar responsiveness and their learning abilities20. In order to catch only nectar foragers, an artificial food source filled with a sucrose solution can be installed near the hive and animals can be collected there. Pollen foragers returning to the hive can be identified by the pollen baskets on their hind legs20.</li>
<li>Fixing and handling: Extended cooling of bees on ice should be avoided because it might affect their ability to learn and to survive until the end of the experiment. Tarsi have to be fixed carefully within the tube to avoid accidental stimulation with sucrose solution. This is important, because bees have been shown to learn by tarsal stimulation with succrose solution3,21.</li>
<li>Olfactory conditioning: The number of conditioning trials, the inter-trial interval, and the test time point can be varied depending on the focus of the study3,22.</li>
<li>Feeding: The satiation level of bees is known to affect their learning performance and memory retention3,23. This has to be taken into account when planning the experiments.</li>
<li>Injection: Air sacs are located in the honeybee thorax17. These vital parts of the respiratory system are necessary for survival. Thus care should be taken not to damage the air sacs during injection. The release of air bubbles indicates damage to the air sacs.</li>
</ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Syringe Omnifix 20 ml</td>
<td>&nbsp;</td>
<td>B.Braun, Melsungen, Germany </td>
<td>4616200 V</td>
<td>&nbsp;</td>
<tr>
<td>White refined household sugar</td>
<td>&nbsp;</td>
<td>Kaisers Supermarket, Berlin, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Clove oil</td>
<td>&nbsp;</td>
<td>Bombastus, Freital, Germany </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Multipette plus</td>
<td>&nbsp;</td>
<td>Eppendorf, Hamburg, Germany</td>
<td>4981 000.019</td>
<td>&nbsp;</td>
<tr>
<td>Rack for 30 bee tubes</td>
<td>&nbsp;</td>
<td>Self-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic tubes</td>
<td>&nbsp;</td>
<td>Self-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>UV light-permeable plexiglass pyramid</td>
<td>&nbsp;</td>
<td>Self-made</td>
<td>&nbsp;</td>
<td>height = 30cm, 
apex 3.5 x 3.5cm, 
base 18x18cm</td>
</tr>
<tr>
<td>Sticky tape</td>
<td>&nbsp;</td>
<td>Tesa, Hamburg, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Capillator sticks 1-5&mu;l</td>
<td>&nbsp;</td>
<td>Selzer, Driedorf-Roth, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Capillator tips 1-5&mu;l</td>
<td>&nbsp;</td>
<td>Selzer, Driedorf-Roth, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Filter paper 0.16-0.2 mm thick</td>
<td>&nbsp;</td>
<td>Macherey-Nagel, D&uuml;ren, Germany</td>
<td>MN 616 or MN615</td>
<td>Adjusted to circular pieces with a 1.3 cm diameter</td>
</tr>
<tr>
<td>Disposable hypodermic needle</td>
<td>&nbsp;</td>
<td>B. Braun, Melsungen, Germany</td>
<td>4657683</td>
<td>0.45 mm diameter</td>
</tr>		</tbody>
		</table>
		</div>

behavioural pharmacology in classical conditioning of the proboscis extension response in honeybees (apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Watch this Scientific Journal Video about Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera at JoVE.com

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

behavioural-pharmacology-classical-conditioning-proboscis-extension

Research

JoVE Journal

Neuroscience

19.3K vistas.  Freie Universität Berlin. Se demuestra cómo implementar un método de farmacología del comportamiento en un paradigma de condicionamiento apetitivo olfativo de las abejas (Apis mellifera) mediante la aplicación sistémica de fármacos. Este método permite la investigación de los mecanismos de aprendizaje subyacente y formación de la memoria de una manera sencilla y fiable.

Video: Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...