Name: in vivo ca2+- imaging of mushroom body neurons during olfactory learning in the honey bee
Uploaded: 2009-08-18T13:00:00
Description: Watch this Scientific Journal Video about In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee at JoVE.com

This work is funded by the DFG.

Bee Preparation and Back-fill
<ol>
<li>Catch honey bee foragers at hive and chill on ice for immobilization.</li>
<li>Mount in Plexiglas recording chambers3. Fix eyes and thorax to the walls of the recording chamber with low melting point hard wax.</li>
<li>Pull glass capillaries as commonly used for electrophysiology and break at the tip to obtain a tip diameter of ca. 10&mu;m. Cover tip of the capillary with dye paste. Dye paste consists of a 10:1 mixture of Fura-2 dextran and lysine fixable tetramethylrho...

<ol>
	<li>Joerges, J., K&#252;ttner, A., Galizia, C. 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=Representations+of+odors+and+odor+mixtures+visualized+in+the+honeybee+brain.">Representations of odors and odor mixtures visualized in the honeybee brain.</a> Nature. 387 (6630), 285-288 (1997).</li><li>Rehder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+honeybee's+proboscis+reflex+by+electromyiographic+recordings.">Quantification of the honeybee's proboscis reflex by electromyiographic recordings.</a> J. Insect Physiol. 33, 501-507 (1987).</li><li>Galizia, C. G., Vetter, R. S., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Methods+for+Analyzing+Odor-Evoked+Activity+in+the+Insect+Brain.">Optical Methods for Analyzing Odor-Evoked Activity in the Insect Brain.</a> Methods in Insect Sensory Neuroscience. , 345-388 (2004).</li><li>Mauelshagen, J. <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+olfactory+learning+paradigms+in+an+identified+neuron+in+the+honeybee+brain.">Neural correlates of olfactory learning paradigms in an identified neuron in the honeybee brain.</a> J Neurophysiol. 69 (2), 609-625 (1993).</li><li>Kuwabara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bildung+des+bedingten+Reflexes+von+Pavlovs+Typus+bei+der+Honigbiene+Apis+mellifica,+Hokaido+Univ.">Bildung des bedingten Reflexes von Pavlovs Typus bei der Honigbiene Apis mellifica, Hokaido Univ.</a> Zool. J. Sci. 13, 458-464 (1957).</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> Journal of Comparative Psychology. 97 (2), 107-119 (1983).</li></ol>

In this presentation we have gone through all the steps to investigate in vivo calcium signals in the honeybee. We applied this technique to mushroom body neurons, but imaging may be done on all neurons for which a staining technique can be established. We have focused on neurons in the olfactory pathway of the honey bee. The experiments are all performed in a darkroom. The set up is illuminated with a long wave light which lies out of the optical perception range of the bee and does not excite the dyes. It is...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>low melting point hard wax Deiberit 502</td>
<td>&nbsp;</td>
<td>Dr. B&ouml;hme & Sch&ouml;ps Dental GmbH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FURA-2 dextran potassium salt, 10 000 MW</td>
<td>&nbsp;</td>
<td>Invitrogen/Molecular Probes</td>
<td>F-3029</td>
<td>Protect from light.</td>
</tr>
<tr>
<td>tetramethylrhodamine dextran 10 000 MW</td>
<td>&nbsp;</td>
<td>Invitrogen/Molecular Probes</td>
<td>D-3312</td>
<td>Protect from light.</td>
</tr>
<tr>
<td>n-eicosane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>21, 927-4</td>
<td>&nbsp;</td>
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<td>Kwik Sil Adhesive</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>KWIK SIL</td>
<td>&nbsp;</td>
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<td>Imaging Set-up</td>
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<td>TILL Photonics</td>
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<td>CCD camera</td>
<td>&nbsp;</td>
<td>Imago</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>CED</td>
<td>&nbsp;</td>
<td>Texas Instruments</td>
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in vivo ca2+- imaging of mushroom body neurons during olfactory learning in the honey bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

Watch this Scientific Journal Video about In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee at JoVE.com

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

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