The overall goal of the following experiment is to demonstrate the ease of recording olfactory responses from the insect nervous system. This is achieved by restraining a live moth and hooking electrodes to the antenna or an individual neuron to record odorant induced responses. As a first step, a saline filled glass electrode is impaled in the eye as ground as a second step, and any of the restrained moths are inserted into a saline filled glass electrode, which enables recordings of electrical responses in the form of an antenna gram for recording single unit responses from an olfactory receptor neuron.
The recording electrode is introduced at the base of a sun. Next, the antenna signal is amplified and fed onto an analog digital converter in order to record and save signals. Results are obtained that show dose dependent, odorant induced responses based on the recorded electrical signal in the form of an EAG signal or a spike.
We developed a new method of recording the Enterogram from the restrained life moth of Naval orange one. The main advantage of this technique is the preparation last for long time, and the same moth can be used multiple times to record multiple responses, and the same moth can also be used to record single unit responses from an individual sensor from the antenna. We focus on the naval orange one because it's an important agricultural place in California.
However, this method can be used for other epi dotter insects, not only to address fundamental questions in old function, but also for the development of new attractants. This study of the moth olfactory system uses the naval orange worm. Ullo Ella.
The insect colony is maintained on a wheat brand diet in environmental chambers at approximately 28 degrees Celsius, approximately 75%relative humidity, and under a 16 to eight hour light to dark photo regime, create containers for newly emerged moths by placing up to 10 layers of water soaked paper towels in a plastic box. Collect and sex the emerging moths daily, then transfer them to the plastic box. Cover the box with a perforated cover to allow air circulation to prepare for the electrophysiological recordings.
First, make a holder for the moth by cutting a 200 microliter pipette tip to have a tip diameter of about two millimeters. For EAG or SSR, select a moth that is two to four days post emergence. Insert the moth into the pipette from the end opposite the tip using a humidified tissue paper stub, gently push the moth towards the pipette tip.
Continue pushing the moth until the antennae and part of the head protrude from the tip immobilize the exposed head by covering the head with a non-drying clay, leaving a small part of one eye and two antennae exposed. Place the moth preparation on the platform of the electrogram microm manipulator between the two electrode holders using glass electrodes with tip diameters of less than one micron. Backfill the electrodes with centum limb ringer.
Looking through the microscope impale the exposed area of the eye with one electrode. This serves as the reference electrode. The next step is to place recording electrodes on the antennae.
The antenna of the male naval orangeworm moth is multis segmented, and each segment is adorned with a large number of hair-like structures. The illa, the inset is a scanning electron micrograph showing details of the illa to facilitate recording from the antennae. Cut off the distal one to two tenal segments.
Next, insert both antennae into the recording electrode. Alternatively, for single sens recordings, insert the recording electrode at the base of a sens. The action potentials from the sens are amplified by a high impedance pre amplifier and fed into a USB intelligent digital analog converter to be analyzed offline with autos spike software.
The next step is to prepare the chemical stimulus using ZZ 1113 HEXA deca Now, which is a major constituent of the female pheromone blend from the naval orange worm, dilute the chemical with hexane to make DIC dilutions. Load a 10 microliter Eloqua of a stimulus solution onto a watman filter paper. Strip evaporate the solvent by gently shaking it for 10 seconds.
Under a fume hood, place the strip in a five milliliter polypropylene syringe, hexane alone, and an empty syringe service controls the preparation. EAG or SSR is held in a humidified airstream delivered by the syntax stimulus controller at 20 milliliters per second. Attach the syringe containing the chemical stimulus to the Airstream controller.
Record two seconds of baseline electrical signals. Then add a four milliliters per second stimulus pulse to the Airstream for 500 milliseconds. Continue recording electrical signals for eight more seconds.
Allow at least one minute between stimulations. This figure shows a robust dose dependent response from the antennae of a restrained male moth. Stimulated with the female pheromone component, CZ 1113 hexaco.
The response to hexane. The vehicle control is shown by the blue line successively. Larger doses of the pheromone generate successively larger electrogram recordings.
This figure shows the extracellular single unit recordings from a tric andum in response to stimulation from the female pheromone component. These recordings show a dose-dependent excitatory response. Once mastered, this technique can be performed in a couple of hours If done properly.
When using this method, make sure to avoid the contamination and use chemicals as pure as possible.
