The overall goal of this procedure is to study the chemo sensory physiology of individual peg illa on a scorpion pectin. This is accomplished by first immobilizing, an anesthetized scorpion ventral side up on a glass slide. Using modeling clay to set up requires submerging the pectins in a custom made oil-filled chamber and making electrodes filled with a chemical stimulant.
Chemos sensory responses are recorded at individual illa on the pectin, which ultimately produce similar response profiles to aqueous chemo stimulants. We Used to record from the basis of illa while blowing volatile stimulants across the pores. The main advantage of the tip recording technique is that it allows the stimulation to be isolated to one pig centum without affecting the activity of its neighbors.
This method can help us answer key questions in the field of chemo sensory biology, for example, why do animals use so many repeated silla in chemical detection and perception? Scorpions might not seem like the safest animals to study, however, not all species are dangerous or difficult to deal with. For example, the desert grassland scorpions are only four to five centimeters long are easy to maintain in the lab and rarely sting if handled properly.
If stung, the venom artificially triggers your sensory neurons, some of which transmit pain information. The sensation usually subsides in about 15 minutes. Scorpions are hardy subjects and can endure multiple days of experimentation, often being returned to their habitations.
When the experiment is complete to immobilize the scorpion, place it in a pre chilled glass container. Then place the jar containing the scorpion into a freezing environment for a minimum of two minutes and remove it from the freezer when the animal is motionless. Now place the immobilized scorpion ventral side up on a microscope slide.
Secure the stinger tail legs and pedi pals with moldable clay. The scorpion's pectins are paired comb like structures near the sex organs and are unique to these animals. A chamber must be made to contain a single pectin without removing it from the animal.
First, make a platform for the pectin to fit onto a glass cover slip cut to approximately five millimeters by 18 millimeters. Usually serves well. Second adhere double-sided adhesive tape to the platform and trim the tape to fit if necessary.
Third, melt some 100%beeswax chips in a glass dish using a hot plate. Now dip three of the platform's four edges into melted beeswax to a depth of three to four millimeters. The cooled wax serves as a wall to make a chamber.
Now, place the unwed edge of the platform just posterior to the point of pectal Insertion on the animal. Stabilize the platform by gently pressing its right and left peripheral edges against the clay used to restrain the animal's legs using fine tipped forceps. Carefully hold one of the pectins along the pectal spine and gently bring it out from underneath the cover glass and lower it onto the adhesive platform floor.
Stick the pectin down by carefully applying pressure to the pectal spine. Complete the chamber by using a preheated metal spatula to apply melted wax to the unwed edge of the platform. The animal is now almost prepared for doing electrophysiology recordings.
Now is an easy time to make an indifferent electrode connection. Insert a silver wire between two tail segments into the scorpion's hemolymph. Press some of the wire that exits the animal into the clay to secure it and leave enough length to reach the amplifier.
Now set the preparation aside for at least an hour before making recordings to allow the preparation to settle for recordings. Use a micro pipette puller to make pipettes with an inner diameter of about two microns larger than the sensor pore diameter, which varies between animals. One hour before recording, fill the pectin chamber with five microliters of mineral oil.
Then position the animal beneath the high powered microscope with long working distance objectives and epi illumination to chemically stimulate peg illa. Load the stimulant pipette with aqueous tastes mixed in an electrolytic solution. Using a micro pipette filler, then attach the loaded stimulant pipette to a micro manipulator.
Now insert the recording electrode into the open end of the stimulant pipette. It is important to ensure contact between the metal electrode and electrolytic stimulant solution. Connect leads of the recording and indifferent electrodes to a differential amplifier.
The amplifier is connected to an oscilloscope to visualize electrical activity and to a speaker to audibly detect electrical change. The setup is now ready for taking recordings. Using the micro manipulator carefully lower the pipette onto a sensor pore.
A change to higher frequencies will be heard when electrical contact is made between the pipette electrolyte and the fluid within the peg sens. The distinct popping of neural action potentials can be heard at this time as well. The duration of chemical stimulation per perpetual sens can vary from as much as 30 minutes to as little as one second.
Generally, one pipette can sample more than 30 pegs. Replace clogged pipettes with clean pipettes. Pipettes are easy to prepare and clogging is not uncommon, so always keep extra pipettes at the ready.
It is possible to record from a single animal for several hours in a single recording session before having to change the mineral oil. A single animal can be kept in place in the recording rig for multiple recording sessions over several successive days. For subsequent sessions, reuse the pectal chamber, but replace the mineral oil.
Just remove all the old oil with a pipette and add a fresh drop. This helps minimize the presence of residual stimulant, which may have leaked out of the stimulant pipette during the recording session. Here are representative tip recordings to different stimulants.
This trace shows a relatively consistent response to 0.01 molar potassium chloride. This peg sends response to 0.1 molar citric acid begins much more intensely then tapers down in intensity within a fraction of a second, followed by some diminishing in frequency. In a typical response to 30%ethanol, the trace shows bursts with multiple spikes between short gaps of inactivity when comparing tip recordings of different sensor responses to the same stimulant, like 0.1 molar citric acid.
The responses are very similar. While attempting this procedure, it's important to remember to fill the pipette with a water-based stimulant, which doesn't mix with the oil medium and allows you to chemically stimulate one lum at a time. After watching this video, you should have a good understanding of how to controllably stimulate individual CIN on a scorpion pectin using mineral oil as a medium.
