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

The overall goal of these procedures is to reliable and robustly administer pharmacological agents to the central nervous system of honeybees or similar insects. These methods can help answer key questions in the fields of sociobiology, neuroethology, and apiculture such as exploring the neuromechanisms of social behavior. The main advantage of these techniques is that it makes it possible to study neurobiology in non-model organisms, such as the honeybee, where genetic tools are limited currently.

Demontrating the procedure will be Jenny Plath, a graduate student from the Neuroethology Lab at Macquarie University. Drugs can be administered orally or by injection. Injections can be made into the thorax, into the ocellus, or into the ocellar tract.

Ocellus injections deliver drugs to the whole head and ocellar tract injections target specific regions of the brain. For all injections, make a microscalpel by breaking off the corner of a double-edged razor blade with a blade holder. Attach the blade fragment to a blade holder so that it makes a nice blade with a sharp endpoint.

For a thoracic injection dissect a two millimeter hole just above the scutellum, next to the posterior wing process of a bee's thorax. Avoid cutting too deep, as this might injure the flight muscles, and be careful to avoid the wing hinges. Ideally, only cut three sides so that the flap of the cuticle can later be folded back to close the site of the injury.

Now inject the drug into the thorax. Then move the cuticle flap back into over the hole and after ten hours it will reattach and seal. To access the brain, lock the head of a harnessed bee in place by filling the neck crevice with hand-warmed dental wax in order to avoid damaging antennal olfactory receptors or other cells that may be important to the bee's behavior.

The antenna may also be locked with dental wax. Then using the microscalpel, carefully remove the lens of the median ocellus by inserting the tip of the microscalpel under the lens and gently breaking the lens free from the head capsule. For the drug to be taken into the whole head capsule carefully pipette the drug onto the ocellus hole and wait for it to soak in.

Alternatively, subregions of the brain can be targeted by injection through the ocellar tract, for example, by injecting 250 nanoliters of drug in a depth of 50 microns to target brain structures adjacent to the ocellus. Then remove the dental wax and allow the bee to rest for a while before continuing the experimental procedure. To prepare injection needles pull one millimeter glass tubes to produce an approximately 0.5 centimeter long tip.

Next, under a stereo microscope break the tips to make and approximately 10 to 15 micron outer diameter opening. This can be measured using a scale on a graticule. Now load an injection needle with the drug solution.

Then at the injection system, attach the loaded injection needle to the micromanipulator. Test the injection system in mineral oil and calibrate it to deliver a 0.5 to two nanoliter bolus depending on the size of the brain structure targeted. Using a graticule, measure the diameter of the droplets to calculate their volume.

To fix the head of a harnessed bee use soft dental wax, as before. Then cut an opening into the anterior part of the head capsule using a microscalpel with three cuts:one just below the median ocellus;one at the border of the right or left eye;and one above the antenna stems. Use a piece of dental wax to hold the opened flap in place.

Next carefully push aside the glands and trachea using fine forceps and make a small rupture into the neurilemma above the targeted brain structure. Now insert the tip of the glass pipette into the desired brain region, ideally, perpendicular to the brain surface into the required depth to reach the targeted structure. Then inject the drug bolus.

When targeting paired brain regions make one injection into each hemisphere to affect both hemispheres. If a fluorescent dye is injected with the bolus the superficial brain regions can be viewed using fluorescent light. Other regions can be checked later using confocal microscopy.

After the injection replace the flap of the cuticle and seal the cut by melting a crystal of eicosane that is approximately one millimeter in diameter over the cuticle using a thin wire attached to a microsoldering iron. This will greatly reduce mortality. To administer a drug as volatile as from a heated nichrome filament, prepare it dissolved in pure ethanol.

To carefully drill holes in the lid of a 50 millimeter tube feed the two wires through the holes. To create a filament, wind up nichrome wire tightly around a nail, attach the nichrome wire to two electrical wires, and then remove the nail. Afterwards, secure the wires and seal the holes with silicone.

Now attach the wires to a power source. And use a thermocouple to measure the temperature of the filament. To calibrate the device, carefully pipette two microliters of drug solution onto the filament and let the ethanol completely evaporate at room temperature.

Vary the voltage in current so the drug bolus volatilizes within ten seconds of applying power. The drug should not be destroyed by thermal decomposition. Precipitate can usually be seen on the dry filament if inspected carefully.

To deliver the drugs to a bee, load a free-flying or harnessed bee into the tube and carefully attach the lid with the wires out. Then turn on the power source for 10 seconds and wait 50 more seconds for the volatilized compound to be fully taken up by the bee before removing the bee. Specific effects on brain processes can be easily obtained following thorax injection.

For example, blockers of transcription were administered using this technique to identify phases of memory that required gene expression. Selective and time-dependent affects on memory tasks were obtained. Diffusion of molecules into the head hemolymph leads to quick, dose-dependent effects arrived at by ocellus injection.

At certain dose concentrations, diffusely-concentrated allatostatins reduced performance in an olfactory learning assay. To test the spatial and temporal properties of specific brain regions, harnessed bees were trained in an olfactory PER conditioning paradigm and then injected with procane in the mushroom body calices, or, vertical lobes. When bees were then tested for recall, performance was only impaired in bees injected bilaterally.

In an arena with multiple feeders, bees were gently captured as they were about to alight from a feeder with one molar sucrose. The captured bees were treated with 100 micrograms of volatilized freebase cocaine, which resulted in increased foraging at half molar feeders, but not two molar feeders. After watching this video, you should have a good understanding of how to administer drugs to free-flying or restrained bees either by injection or volatilization.

Once mastered, these techniques can easily be done on a large number of bees in a couple of hours. Following this procedure, other methods, like proboscis extension conditioning, can be performed so we can answer additional questions about the neurobiology of learning and memory.