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.

This procedure begins with fixing the bee into a recording chamber. A piece of cuticle is removed from the head capsule to stain selected structures in the brain. Calcium signals in the stained neurons are recorded in vivo using a wide field imaging setup.

During imaging, the bee can be stimulated with odors or sucrose delivery to the antenna. This way, the bee may be conditioned to extend the proboscis in response to an odor. The proboscis extension response is monitored using electromyogram recordings from the M 17 muscle.

After the imaging experiment, the brain is dissected for a closer investigation of the stained structures using a confocal microscope. Hi, I'm Melanie from the Lab of rdo Mansel. Hi, I'm Anya also from the Mansel Lab.

Today we will show you a procedure for in vivo calcium imaging and the honeybee mushroom body. We use this procedure in our laboratory to study olfactory coding and learning related plasticity in the honeybee brain. So let's get started.

Start by catching a honeybee forager at the hive entrance, and then immobilize it by chilling it on ice. Next, mount the bee onto a plexiglass recording chamber. Use low melting point hard wax to fix the eyes and thorax onto the wall.

All break a glass capillary at the tip to obtain a diameter of 10 microns. Cover the tip of the capillary with d paste consisting of a 10 to one mixture of URA two dextrin and fixable Rodin dextrin. The prepared capillary will be used for staining.

To begin the staining procedure, first immobilize the antennae with an koane. Open the head capsule above the brain by removing a piece of cuticle and pushing the glands and trachea to the sides, allowing access to the mushroom body. Use a piece of paper to absorb the hemo lymph inside the head capsule.

To stain mushroom body neurons, inject the capillary into either the soma or dendritic region of the neurons. Then restore the cuticle piece on the head capsule and loosen the antenna. Finally, feed the bee with 30%sucrose solution and store it in a styrofoam box covered with a wet kitchen towel for at least four hours or overnight at 20 degrees Celsius.

With B preparation complete, we can begin in vivo imaging. To begin, use a sponge that is attached to the recording chamber to prevent the bee from moving by, pressing it up against the abdomen. Now fix the antennas of the bee with an ioane to prevent the brain from moving due to the pumping of the esophagus.

Cut a small incision into the cuticle above the labrum and carefully pull out the esophagus and its surrounding solid structures and cover them with two components silicone. Next, use a needle to make holes for the insertion of wire electrodes used to record electrograms from the protractor muscle of the labium. Remove the cuticle piece, trachea and glands above the brain.

Use a piece of paper to absorb the hemo inside the head capsule to record electrograms from M 17. Inject a copper wire into the muscle close to the mouth parts. Inject a ground electrode into the eye.

Now fill the head capsule with two component silicone. Make sure that the brain is completely covered. Place the bee on the microscope stage and place a drop of water on the surface of the silicone.

Immerse the dip objective of the microscope into the droplet, and then focus on stage neurons. With the preparation for in vivo imaging complete, we can now describe odor stimulation and signal recording. To deliver odor stimuli to the bee, we use a computer controlled custom-built olfactometer to dilute odorants into a constant airstream.

Directed at the antenna odor stimulation consists of a three second pulse of odor saturated air. For calcium imaging, we use a til photonics imaging setup mounted on a zes fluorescent microscope to record images at room temperature with a sampling rate of five hertz. Calcium signals are recorded through an X 60 0.9 W Olympus dip Objective with Ango CCD camera, each measurement lasts 10 seconds.

The muscle potentials are amplified with a grass amplifier and recorded and digitized with an analog digital converter. Recordings from M 17 are used to monitor behavioral responses related to learning with signal recording complete, we can now move on to morphological analysis and reconstruction. Because both dyes used during backfill have the same molecular weight.

They're co-located in the neurons. Widefield imaging has limited spatial resolution, so we use confocal scanning microscopy to investigate the stained structures. After the experiment, first dissect the brain and fixate it overnight in 4%formaldehyde at four degrees Celsius the next day, rinse it in PBS and then dehydrate it in ethanol steps 10 minutes in 50%70%90%99%and two times 10 minutes in 100%Place the brain on a grooved object slide with a drop of methyl salicylate cover, slip it and then place it on the microscope stage of a confocal scanning microscope.

Scan the brain with an air objective lens and a 1.1 to 1.2 digital zoom at four micrometer optical sections. Here we see the response of mushroom body extrinsic neurons to odor stimulation of the antennae recorded through a 60 x objective and visualized in false colors. The red square to the left represents the persistence of the odor stimulus.

After the experiment, the brain is dissected and the stain structures are investigated in greater detail using a confocal microscope at 20 x magnification. So we have just shown you how to image mushroom body neurons in the honeybee during Olfactory learning. When doing this procedure, it's important to remember that experiments involving light sensitive dyes have to be performed in the dark.

So that's it. Thanks for watching and Good luck with your experiments. Bye bye.