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

Summary

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.

Abstract

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, Küttner 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.

Protocol

Bee Preparation and Back-fill

1. Catch honey bee foragers at hive and chill on ice for immobilization.
2. Mount in Plexiglas recording chambers³. Fix eyes and thorax to the walls of the recording chamber with low melting point hard wax.
3. Pull glass capillaries as commonly used for electrophysiology and break at the tip to obtain a tip diameter of ca. 10μm. Cover tip of the capillary with dye paste. Dye paste consists of a 10:1 mixture of Fura-2 dextran and lysine fixable tetramethylrhodamine dextran.
4. For the staining procedure immobilize antennae with minutiae or n-eicosane. Remove a piece of cuticle above the brain and push g....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
low melting point hard wax Deiberit 502	Dr. Böhme Schöps Dental GmbH
FURA-2 dextran potassium salt, 10 000 MW	Invitrogen	F-3029	Protect from light.
tetramethylrhodamine dextran 10 000 MW	Invitrogen	D-3312	Protect from light.
n-eicosane	Sigma-Aldrich	21, 927-4
Kwik Sil Adhesive	World Precision Instruments, Inc.	KWIK SIL
Imaging Set-up	TILL Photonics
CCD camera	Imago
CED	Texas Instruments