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

The overall goal of this procedure is to study olfactory coating and memory formation using long-term access to two independent neuronal processing stages within behaving honeybees. This is accomplished by first assembling low-cost multi-channel electrodes from insulated copper micro wires and custom made holders. The next step is to prepare a living honeybee by removing a section of its head capsule to insert two electrodes treated with a fluorescent dye into the exposed brain.

The third step is to record from the two independent neuro pills or tracts in the olfactory pathway, while stimulating with floral odors and pheromones. Later, after dissecting the brain, a different fluorescent tracer is injected into the antenna lobe to backfill the target neurons. The backfilled tracks and the electrode tracings are then visualized to produce a three-dimensional reconstruction of the neuronal architecture.

Ultimately, relationships in odor coating between the two neuron populations at different processing levels like the antenna lobe and the mushroom body can be better understood using these techniques. So the main advantages of this technique of existing methods is that our multichannel electrodes are very small and can be used in combination, meaning that we can record simultaneously at different areas of the brain after being anchored. The flexible wires allow a stable recalling for several hours in behaving animals.

This method can help answer key questions in the field of neuropathology, such as understanding how animals receive, perceive, and finally compute olf factories in the life from their environment. This double recording technique can provide insight into the underlying mechanism of odor coding and neuronal networks in general. It can also be applied to other model organisms in invertebrate or affection and neurobiology.

Generally, individuals new to this method will struggle because a lot of training is necessary to identify honeybee neurons using only morphological landmarks like the internal lobe or the vertical lobe of the mushroom body. For this protocol, build an electrode adapter to fit a standard electrode interface board. First solder three short pieces of insulated wires to an 18 pin connector.

Then cut a piece of plexiglass plate and insert a shallow groove along the longest side. Next, screw soldering lugs into the plexiglass plate and glue the plate onto the top of the connector. Then connect the base to the lugs using the three short wires.

Next position a glass capillary into the groove of the plexiglass plate. The capillary should be able to slide and be secured by a screw Using a minutian pin, extend the glass capillary outward about five millimeters. The capillary and pin will be used to support and stabilize the electrode microwires.

Next, position three microwires. With the help of adhesive tape for better access, the glass capillary can be removed using a 12 volt soldering needle glue. The wires together with LMP dental wax do not glue the last third of the wires.

Now connect this multichannel micro wire assembly to the electrode adapter. Position the extended glass capillary parallel to the micro wires. Secure this attachment with dental wax along the overlapping glass capillary and the minutian pin.

Then trim the electrode wires leaving two to three centimeters extending from the minutian pin and two to three centimeters extending from the electrode end. Next, secure the capillary into the electrode adapter and use a screw to stabilize everything in place. At 360 degrees Celsius.

Solder the ends of the wires perpendicularly to the lugs. Then check that there is a sufficiently strong electrical contact by checking that their impedance is about 300 kilo ohms at one kilohertz. Now connect the channels of the second electrode adapter to the master electrode and solder the reference and muscle electrodes to the master electrodes base.

Finally, mount the master electrode to the interface board of the head stage. Fix the second electrode on a separate adapter after obtaining the honeybees chill one on ice until it is immobilized. Next, fix the bee in a standard plexiglass holder with the head exposed.

Apply heated dental wax to the base of the eyes and the junction between the head and thorax, thus securing the head. Continue using the wax to immobilize the scape eye of the antennae on the head capsule. Avoid applying wax to the fagel, which needs to point forward.

The Pro Bois should be free from impingement. Check that it can move. Now shave the head capsule to improve accessibility.

Then feed the bee all the 30%sucrose it will consume. This ensures good preparation, viability and tissue moisture. Now using a micro scalpel, cut vertically along the eye border and horizontally above the antenna bases.

Continue cutting under the O cell eye and remove loose cuticle. The hypo pharyngeal glands should be moved aside, as should the trachea. This will clear the path for inserting the electrode.

Begin with inserting the reference electrode, a 35 micron silver wire. First, make a small cut in the ipsilateral compound eye. Then tuck the electrode through it.

Next, insert the second wire into the muscle projection region below the lateral O cell eye. To later visualize the electrodes positions, dip the electrodes in a solution of 0.5 molar potassium chloride, and 5%Alexa Hydrocyte 4 88 for three minutes. Next, using micro manipulators position the two electrodes into the brain.

Orient yourself by identifying the antenna lobe and the vertical lobe of the mushroom body. Here one electrode is in the lateral antenna lobe tract 180 microns deep and the other in the medial antenna lobe tract. Inserted about 300 microns deep.

Complete the electrodes placements by anchoring them using two components. Silicon cover the entire space above the brain, which will also prevent the tissues from drying out. Then proceed with making recordings to extract unit activity from the extracellular recorded signal.

Use available spike sorting software. Use the differentiated electrode channels. Apply template matching techniques simultaneously on all three electrode channels to detect different waveform combinations that account for single spike events.

And finally, control for proper unit separation. Using principle component analysis on the single spike events of the three recorded differentiated electrode channels. After making recordings carefully remove the silicon and electrodes from the bee.

Next, rinse the brain with bee ringer solution and remove the glands and trachea. Then using a micro pipette inject into the antenna lobe, 5%micro ruby dissolved in one molar potassium acetate. This will provide an anterograde label to the lobe's tracks.

From here forward, perform all steps in maximal darkness. Rinse the brain with bee ringers again, three times, and allow the dye to incubate for 30 to 45 minutes. Immobilize the bee by cooling it in the fridge.

Then isolate the brain. Fix the brain in 4%Para aldehyde in 0.1 molar PBS overnight at four degrees Celsius and with gentle agitation. Follow this with standard protocols for dehydration and clearing After back filling projection neurons in the antenna lobe with micro ruby tracer, a projection view of these neurons was assembled from ortho slices along the Z axis.

The Alexa Hydrocyte 4 88 dye on the two electrodes shows their insertion at the medial antenna lobe tracted, and in the lateral antenna lobe tract. A 3D reconstruction of the stained target cells and the electrode insertion sites was used to make a schematic of the two antenna lobe tracted trajectories simultaneous recording from the two antenna lobe tracts while stimulating the bee with different odor concentrations was used to investigate temporal processing. Activity was assessed in subsequent processing stages.

The antenna lobe projection neurons and the extrinsic neurons principle component analysis of the population vectors illustrates that odor in the projection neurons was prolonged and outlasted the stimulus. By contrast in the extrinsic neuron population, only odor on and odor off types were represented. So in viewing activity with time, the extrinsic neuron population on the right shows odor separation activity slightly before the projection neuron population starts to develop their odor induced activity.

Odor stimulation is marked in gray. Once mastered, it takes about 30 minutes to build an electrode. Preparing the bee and inserting the electrodes into the target regions can be done in another half hour if it is performed properly.

While attempting this procedure, it's important to remember that every step takes a lot of practice and patience until it's mastered. Be especially careful when soaring the electrode wires to their connection points and recording from the desired brain reach. Its required solid knowledge of honeybee brain anatomy After its development.

This technique enabled researchers in the field of neuropathology and insect olfaction to explore the precise temporal relationships between the activity of different neuronal pathways and brain centers in behaving honeybees. After watching this video, you should have a good understanding of how to construct and use multichannel microwire electrodes to record simultaneously from two brain regions and behaving honeybees. You should also be able to visualize the exact locations of your recording electrodes.