The overall goal of this procedure is to identify biologically effective odorants in complex sense through recordings in the bumblebee brain. This is accomplished by first collecting behaviorally effect sense, and second, preparing the gas chromatograph and multichannel recording systems for the experiment. The third step is to prepare and dissect the bumblebee brain for the neural recordings in the antenna of factory lobe.
The experiment is then conducted by inserting the electrodes into the an antenna lobe and measuring responses to puffed volatiles by analysis of the neural activity in response to complex sense and their individual constituents. Biologically active odorants obtained by gas chromatography can be identified. So the main advantage of this technique relative to other techniques like G-C-E-A-G, is the sensitivity of antenna load neurons and the ability to characterize how neurons in the brain respond to specific floral volatiles.
Visual demonstration of this method is critical as a combined electrophysiological and chemical analytical steps are difficult to learn because of the electrophysiological preparation recording technique and the necessary steps for collecting headspace volatiles. Although this technique can provide insights into the olfactory processing of bumblebees, it can also be applied to other systems such as disease factors like mosquitoes or kissing bugs, or crop pests like aphids or moths. For this procedure, have prepared a reference electrode made of tungsten wire on a micro manipulator on a second.
Micro manipulator have a multi-channel electrode, such as a coiled wire, tede, or silicon multi-channel electrode connected to a pre amplifier have a gas chromatograph with an output on a shielded BNC table. The output of the GCs detector is interfaced with the amplifier and data acquisition system such that both the neural and GC signals are synchronized. In this example, the GC carrier gas is helium and is in a DB five GC column.
The GC uses a split list inlet with the temperature set to 200 degrees Celsius and has the flame ionization detector temperature set to 230 degrees Celsius. After the samples are heated in the gc, the effluent from the column is split one to one between the flame ionization detector and the B antenna using a glass y connector. In this example, the GC is used to deliver volatiles collected by dynamic absorption from Mimus Lewis eye flowers, an alpine wildflower native to California.
Alternatively, instead of using the gc, single volatile compounds or mixtures of compounds can be delivered by air pulses. Shot through a separate glass syringe containing a piece of filter paper on which the compounds have been deposited. The air pulses are diverted from a constant Airstream using a solenoid activated valve controlled by a computer.
Begin the preparations by cutting about one centimeter from the end of a one milliliter pipette tip. Place a bumblebee into the base of the pipette tip and gently push it towards the opposite end of the tip until only the head is exposed. Using melted dental wax, make a mold around the exposed head.
The wax must adhere to the compound eyes to completely immobilize the head, but it mustn't contact the antenna if necessary. Use small pins to immobilize the antennae. Next, make a square window like incision into the head capsule using a sharp blade such as a micro scalpel.
Begin cutting directly behind the antenna and adjacent to one of the compound eyes. Cut a straight line between the eyes. Next, cut dorsally until the head capsule curves to the thorax.
Continue the cut to the opposite end of the head capsule. Finally cut back to the start to complete the square. Use forceps to remove the cuticle.
The cuticle adjacent to the antenna must be removed, or it will impede the electrode as soon as the brain is exposed. Immediately super fuse the tissue with insect saline. Make sure the antenna lobes are well exposed.
Then carefully use a pair of very fine forceps to remove the perineural sheath immediately above the an antenna lobes. Be very careful not to puncture the bee's brain with the forceps. The bee preparation is now ready for electrophysiological recording.
Begin by attaching the prepared bee to a clamp that is fixed to a magnetic base. Keep it super fused with insect saline delivered from a reservoir with a flow controller. Now insert the reference electrode into the contralateral brain tissue.
Next, insert the multichannel electrode into the an antenna lobes of the bee. When the electrodes are in place. Wait 30 to 60 minutes for the recordings to stabilize.
During this time, prepare to the extracellular spikes from neurons by also thresholding. The individual recording channels Manual thresholding may be required for some channels due to a noise in the recording channels. Next, verify that the temperature ramp of the GC run is correct.
In this example, the temperature starts at 50 degrees Celsius for four minutes and then increases 10 degrees per minute to a final temperature of 220 degrees Celsius, or it is held for six minutes. When the waveform shapes of the units in the recording channels have become consistent, the neural recordings are considered stable. Once stabilized, load the syringe with the floral headspace odor and inject the odor into the head injection port of the gc.
Then immediately begin recording from the electrodes after the GC run has finished. Let the preparation rest for five to 15 minutes. Then either inject another sample into the GC or stimulate the preparation using single volatile compounds or mixtures of compounds.
If at any point the spontaneous activity suddenly stops or changes, check the saline drip and let the preparation rest for 15 minutes. Then if the activity does not regain its previous level, discard the preparation. After the experiment, fix the brain with the probes still in the tissue, then excise the brain, clear the fixative and dehydrate the brain.
The locations of the recording channels can then be identified by confocal microscopy from the mimus Luci floral scent. The total number of volatiles are looting through. The GC is typically between 60 and 70 compounds.
The scent of Mimus Luci is predominantly composed of mono terpenoids, including beta myosin and alpha pinine. The remainder of the scent is composed of six carbon volatiles. Several preparations were stimulated with three microliters of the Mimus Lewis eye extract.
This recording from one of the eight neural units recorded by the multichannel electrode shows how selective a neuron compete to specific volatiles. In this example, dine is that compound shown. Here are the responses of eight neural units.
About half of the ensemble was unresponsive. This is surprising given the diversity of volatile compounds alluded from the floral headspace, but this is consistent between preparations and is reported by other studies. The normalized firing rate of the responsive units shows that there is a strong selectivity for a group of odorants.
The odorants highlighted by the arrows elicited the most robust responses. These are demine and trans beta osain. So after watching this video, you should have a good understanding of how to prepare your insect for the experiment and then conduct the experiment using gas chromatography with multi-channel recording.
The goal then is to identify putative behaviorally effective odorants in a complex bouquet. Once mastered, this technique can be performed in two to three hours if done properly Following this procedure. Other methods like behavioral testing with the identified odorants can be performed in order to fully determine the efficacy of the volatiles in mediating the behavioral response of the insect.
