The overall goal of this procedure is to record odor revoked multi-unit neural activity from the first three signal processing centers of the insect olfactory pathway. This is accomplished by first restraining a locust in a custom designed chamber and stabilizing one of its antennae for electrophysiological recordings. The second step is to fabricate a glass micro electrode and insert it at the base of a sun to monitor the odor evoked responses.
Next to record neural activity from the locust brain. A multi-step dissection procedure is followed to expose the neural tissue, which processes the input from the olfactory sensory neurons. The final step involves appropriate placement of the multi electrode arrays to record ensemble neural activity from the locust and antenna lobe and the mushroom body.
Ultimately, the extracellular recording methods demonstrated here are used to characterize odor evoked responses at the first three processing centers along the insect olfactory pathway. Though this method has been used to characterize the odor evoked neuro response in a relatively simple invertebrate olfactory system, it can also be adapted to examine the ensemble activity in other neural systems as well. Visual demonstration of this method is critical because some key aspects of this procedure, such as electrode placement and some dissection steps are very hard to learn.
Additionally, the methods are mostly qualitative and different labs have perfected it differently over time. To begin this procedure, select a young adult locust of either sex with fully grown wings, but prior to the mating stage from a crowded colony. Next, restrain the locust by amputating its legs.
Seal the amputation sites with tissue adhesive. Then secure the locust to the custom designed chamber with a small piece of electrical tape draping around its thorax. Under a dissection microscope, make a shallow groove in the wax platform for antenna placement.
Place the antenna into the groove and stabilize it using bati wax at the two ends of the antenna. After that, insert a ground electrode into the thorax. Use Batik wax to seal the incision site and to hold the ground wire in place.
Next, place the stabilized locust antenna under a stereo microscope on a vibration isolation table. Connect the outlet lines of the odor bottles to the odor delivery tube. Position the odor delivery tube within a few centimeters of the antenna delivered odor vapors are removed by a vacuum funnel, placed approximately 10 centimeters behind the locust antenna.
Then fabricate glass electrodes using a burs silicic glass capillary with a micro pipette polar fill the glass electrode with locust saline. Next, place the glass electrode into a micro pipet holder that is attached to a motorized micro manipulator. Make sure that the base of the recording syn is clearly visible under the microscope.
Gently insert the electrode into the base of a syn. Subsequently amplify the signal 10, 000 times. Using an AC amplifier.
Filter the signal between 0.3 to 10.0 kilohertz and acquire it at a 15 kilohertz sampling rate. Using a data acquisition system, when the electrode is appropriately positioned, spontaneous neural activity can be detected. Stimulus evoked responses can be monitored by delivering an odor pulse through the odor delivery tube.
Positioned close to the antenna and tracking the change in the extracellular activity, followed the restraining procedures as described previously. Position the locust in a custom designed chamber. Begin the dissection process by building a wax cup around the locust head.
Construct two wax towers on either side of the locust head, reaching up to the height of the antennae. Next, insert each antenna through a small piece of polyethylene tubing that is tightly fit in a rubber gasket. Then attach the gaskets to the wax towers such that the plastic tubing can freely slide towards or away from the base of the an antennae while preventing the an antennae from moving.
Then under the microscope, start building a wax cup around the locust head to allow saline perfusion during and after the dissection procedure. The wax cup should start just above the mouth parts, encompass both the wax towers and extend beyond the compound eyes. Once the wax cup is fully constructed, attach the base of the an antennae to the bottom end of the plastic tubing using epoxy resin.
This will ensure that the an antennae are held in place even after the surrounding cuticle is removed. Keep the wax cup filled with saline solution from this point onwards. Now, remove the central rectangular region between the two.
An antennae. Subsequently, remove the cuticle in the neighboring regions without disturbing the compound eyes or cuticle at the base of the an antennae. Using fine forceps, gently remove the air sacks and fat bodies surrounding the brain.
The locust brain should now be clearly visible. The brain regions that process the olfactory information are situated between the two. An antennae gently pull the for gut and cut it using fine scissors.
Then remove the wings and make a small incision in the abdomen just above the rectum. Remove the gut by pulling the hind gut with coarse forceps. This step ensures that gut movement does not destabilize the preparation.
To prevent saline leakage. Tie the abdomen just anterior to the incision site with suture threads. Next, use a small platform made of a thin wire loop coated with a fine layer of wax to elevate the brain and stabilize it for electrophysiological.
Recording the brain is protected by a semi-transparent sheath, which must be removed prior to recordings. To weaken the sheath, partially drain the wax cup and carefully spread a small amount of protease over the surface of the brain. After approximately five to 10 seconds of enzyme application, rinse the brain thoroughly with saline.
Then very gently pinch and pull the sheath up with super fine forceps to tear it open and expose the recording sites. In this procedure, place the locust preparation under a stereo microscope on a vibration isolation table. Maintain a constant saline perfusion rate of about 40 milliliters per hour.
Throughout the experiment, use a chloride silver wire immersed in the saline filled wax cup as the ground electrode. Prepare the odor delivery setup similar to the single sensor recording experiment. Prior to the experiment, electroplate the electrodes with gold to achieve the impedances in the 200 to 300 kilo range.
Use a 16 channel silicon probe for principal neuron recordings. Under the microscope. Position the electrode close to the surface of the an antenna lobe and gently inserted into the tissue using a manual microm manipulator in an ideal recording site, extracellular signals will be picked up by multiple recording channels and will have a high signal to noise ratio.
For Kenyon cell recordings, a custom made twisted wire tero is used electroplate the tero following the procedures for electroplating. The silicon probe then place the tero on the surface of the mushroom body. Since the Samara of Kenyan cells are restricted to the superficial layer of this region, both principle neuron and Kenyan cell recordings can be made simultaneously from the same locust preparation.
After identifying the recording location, wait at least 15 minutes to allow stabilization of the electrodes. Then acquire the extracellular signal at 15 kilohertz. Filter it between 0.3 to six kilohertz and amplify it using a 16 channel AC amplifier, both spontaneous and stimulus evoked.
Multi-unit neural activity from projection neurons in the an antenna lobe can be observed. Raw extracellular voltage traces showing responses of an olfactory receptor neuron to two different odors. Two octal and one H ethanol are shown here.
A representative extracellular trace from a multi-unit antenna lobe recording is shown here. The action potentials or spikes of varying amplitudes originating from different principle neurons can be observed in this voltage trace. Note that a four second odor pulse was applied during the time period indicated by the gray box.
An example mushroom body extracellular recording is shown here. Unlike olfactory receptor neurons and principle neurons, Kenyon cells in the insect mushroom body have lower baseline activity and respond to odors in a sparse and selective manner. To isolate single unit responses from these multi-unit recordings, we perform a spike sorting procedure using published software in an offline manner.
Examples of principle neuron and Kenyan cell spike sorting are shown here After watching this video. Should have a good understanding of how to perform extracellular multiunit recordings to characterize ensemble neural activity in the first three olfactory centers along the insert olfactory pathway.
