The overall goal of this procedure is to record complex neural activity in the brain and coordinate it with the video recorded behaviors of freely moving insects. This is accomplished by first manufacturing of flexible recording tetro from extremely fine wire sharpening, testing and coating the tip and material for marking its location and stabilizing the wire in polyethylene tubing. The second step is to insert the recording tede into the brain, stabilize it with acetate and wax, and secure a strain relief loop to the head.
Next neural activity is recorded along with video recording of locomotor behavior. Synchronize the video to the recording by a synchronizing pulse to an LED and the amplifier. The final step is to deposit copper using a continuous electric current and intensify the staining using the Tims method to visualize the recording site.
Ultimately, high density multi-unit extracellular sorting techniques identify neural units while analysis of simultaneously recorded video of the behaviors allows for fine scale correlation of neural activity and behavior. The main advantage of this technique is that it allows us to use existing procedures to record high density extracellular neural activity from an insect's brain while it's performing a complex behavior. It then allows us to identify where in the brain the Tet roads were, and all of this can be done relatively inexpensively.
This technique can help answer key questions in neuropathology by correlating the timing and intensity of neural activity in specific brain regions to the behavior of a freely moving animal. This method can provide insight into sensory motor control, but it can be also applied to any behaviors that require freely moving insect, such as learning orientation or navigation. Generally, individuals new to this message will struggle because the recording TE S are difficult to ate and placing and stabilizing them for recording requires very precise microsurgical techniques.
The visual demonstration of this method is critical. The Tero making and implantation procedures are difficult to learn because they're performed with very small and fragile materials, and you have to take great care so as to not damage the brain tissue. To prepare the Tet Road wires, place two pulled rom wires into a motorized rotating winding device.
Wind the tero in one direction for two minutes at 60 rotations per minute, then unwind it in the opposite direction for 30 seconds. Next, use a heat gun to fuse the wires together, being careful not to touch the wires with the gun. Use three up and down passes from alternating directions with each pass taking about 10 seconds.
Then add a supporting tube made from a 30 centimeter length of polyethylene tubing. Thread the tetra slowly into the supporting tube so that it does not kink. Once the fused end appears at the other end of the tube, pull it through so that there is an equal length of wire at both ends of the guide tube.
Now grab the separate end of each wire with the forceps using the base of the flame of a gas burner. Carefully burn the insulation off of the last two or three millimeters of each wire. Heat the wire until it glows but does not curl.
Next, use a forceps to connect the tero with a male female IC socket adapter that fits the recording device. Put the de insulated end of each wire into a different socket of the adapter. Add a brass pin to help hold the wires in place.
Then use a fine point soldering iron to fill the socket with the melted solder. Next test for proper electrical connections using an impedance meter as described in the accompanying text protocol. Using a separate unused adapter form a small paper mold with holes in the bottom.
For the mail pins, transfer the working adapter into the box with the mail side at the bottom and the pins projecting from the box. After taping the box as described in the text protocol mix fast. Set two part epoxy and pour it into the box.
To secure the adapter and all of the wires, attach the near end of the guide tubing to one side of the box with dental wax, but leave the tubing open such that the wire tero can be pulled through freely at both ends. After the epoxy is hardened and the attire assembly is dry, the next step is to sharpen the tero. Begin by cutting the tip of the tero with a sharp scalpel blade.
Next, polish the tero using a small rotary tool mounted vertically with medium and fine grit sanding discs. Set the rotary tool to spin at moderate speed holding the bundle near its end. With forceps tilt the wire set end to a 45 degree angle relative to the sanding disc.
It is critical that the direction of spin of the sanding discs is away from the shallow angle of the wire ends. Otherwise separation of the wires may occur. Gently touch the bundle to the medium grit sanding disc for one to two seconds, and then touch the fine grit sanding disc for another one to two seconds.
Repeat this three more times. Axially rotating the bundle 90 degrees each time. When finished, use a dissecting microscope to verify that the bundle end has a pointed tip with small amounts of insulation removed from the end of each wire and that there is no fraying After rechecking impedances both between individual wires and between each wire and ground plate.
The tero by putting its tip into a saturated copper sulfate solution Plate the first wire with a current of 2.5 microamps. Using a stimulus isolator, inject the current for one second. Pause for one second and repeat this process three times.
Then plate the remaining three wires in the same way. After again, checking the impedance. Mount the adapter onto the head stage of a multi-channel recording system.
Attach a bent insect pin to a micro manipulator and attach the end of the tero wire to the insect pin with dental wax after anesthetizing and securing a cockroach as described in the text protocol, use a razor blade to cut a small window between the O cell eye after removing the cuticle. Use forceps to remove connective tissues and fat to expose the brain. Then place cockroach saline into the head capsule to cover the brain tissue to de sheath the brain.
Use a fine forceps to gently grab the sheath on top of the brain and use another fine forceps to tear the sheath apart in the wire implanted area. Next, use an insect pin to open a small hole in the head capsule anterior to the brain. Insert a braid of 3 56 micron diameter, insulated copper wires into the hole.
To serve as a reference ground electrode, lower the tip of the tetro to the brain surface with the micro manipulator and position it near the brain. Region of interest. Carefully place two small pieces of two by one millimeter thin acetate sheet slightly larger than the hole in the head capsule anterior and posterior to the tede.
Slowly lower the tede 150 to 250 microns below the brain surface while monitoring the recording quality. Look for moderate to large sized spontaneous extracellular spikes to indicate both an area of interest and general neural tissue viability. When satisfied with the Tero location, move the two pieces of acetate sheet as close to the tetro as possible without touching it.
It's very important to be extremely careful during the dissection and implantation so you don't tear at the wires. Using a small spatula fashioned by flattening the tip of a hypodermic needle, heat the spatula with an alcohol burner. Then put the spatula into dental wax such that there is liquid wax at the tip of the spatula.
Carefully touch the liquid wax to an end of an acetate sheet that is farthest away from the tero, allowing the liquid wax to seal the gap between the acetate sheet and the head cuticle. Continue reheating the spatula and dropping a small amount of liquid wax onto the acetate sheet. Gradually moving towards the tero and eventually anchoring the tero with a small amount of dental wax.
Avoid getting hot wax into the cavity and onto the brain. Next, anchor the reference electrode with wax in a similar manner. Now heat the wax that attaches the tero to the microm manipulator to release the tero from it.
Then loop the tero into the wax on the head to provide a strain relief and cover the strain relief loop with wax. At this point, carefully remove the constraints and transfer the preparation onto a Petri dish, which is sitting on ice. Restrain the preparation dorsal side up with large saddle pins Using a glue gun, attach a wooden stick so that it extends from the pro noum over the abdomen.
Next, attach the tip of the tero tubing to the posterior end of the rod with dental wax. Then anchor the tero and the reference electrode to the anterior end of the rod with dental wax. Then pull the tero from the socket end of the tubing as much as possible, but do not tug on it.
Secure it to the adapter with wax in order to eliminate the chance that the animal may damage the exposed portion of the tero. After removing all the constraints, attach the reference electrode to the tero tubing with dental wax. Establish good neural and video recordings as noted in the text protocol prior to beginning the behavioral experiments for walking experiments, allow the cockroach to explore a plexiglass arena for climbing experiments.
Use a long narrow arena with either a shelf obstacle for the animal to climb over or tunnel under, or else a plexiglass block for the animal to climb. When recording, generate a TTL pulse from the computer using a customized MATLAB command. The TTL pulse generates a timestamp for the recording system and either turns on or off the LED light for stimulation trials.
Trains of current pulses between recording wires can be used to evoke behavior once the experiment has been completed past five seconds of five micro amp, dc current through one of the wire tips and the reference electrode to deposit copper into the brain. To mark the location of the wire tip, raw voltage traces from single electrodes within one tero bundle are shown here. Note the difference of the voltage traces among different electrodes.
Three units were sorted using MLUs. A three dimensional view of the waveform energy as recorded on three of the four electrodes is plotted here.each. is a single threshold event color coded by the cluster it was ultimately assigned to.
This snapshot is taken from a video of a climbing trial above each frame is shown the normalized speed, the height of the cockroach, and the instantaneous firing rate of two sorted units from the first to the current frame times zero indicates the onset of climbing firing rate was normalized from zero to one, and speed and height were normalized from zero to 0.5 for display purposes. The marks below each frame indicate the spikes of the two sorted units within one second of the current frame. The orange line indicates the time covered by each frame and the blue rectangle indicates twice the width of the kernel that was used to calculate the instantaneous firing rate for the current frame.
This recording was made in the right fan shaped body. The firing rate of unit one one increased during climbing and the increase of firing rate proceeded the increase of speed. Unit one two was silent before climbing but started to fire after climbing was initiated.
In this snapshot from the Arena Exploration Trial, the red oval line indicates the shape of the cockroach in that frame and the red dash line indicates the position of the cockroach's center of mass in the previous 10 frames. The turning and forward walking speed as well as the instantaneous firing rate of unit two one are shown here. The spikes of unit two, one within four seconds of the current frame are shown below the frame.
As before the orange line indicates the time covered by each frame and the blue rectangle indicates twice the width of the kernel that was used to calculate the instantaneous firing rate for the current frame. This recording was made in the middle fan body. The position of the cockroach and its body orientation in each frame were extracted using tracking software as described in the text protocol and used to calculate forward and heading speed as well as instantaneous firing rate.
The firing rate of Unit two one increased when the cockroach started to walk and was correlated with walking speed. For many central complex units, increased firing rate was restricted to specific locomotion states. For instance, unit two, one was tuned to forward walking irrespective of turning speed.
The trajectory of the cockroach from the video is shown here. The large black dot indicates the starting point of the cockroach, and each small black dot indicates the position of the cockroach in each frame. The trajectory was color coded to show the instantaneous firing rate of unit two, one ranging from blue to indicate a low firing rate to red to indicate a high firing rate.
In this firing rate map of Unit two one, the X axis is the turning speed, and the Y axis is the forward walking speed. Positive turning speed indicates right turning and negative turning speed indicates left turning. Placement of the electrode is determined by copper deposits visible after the histological procedure as described in the text.
Alternatively, the electrode can be coated in a fluorescent dye and located by confocal microscopy. This is a complex set of techniques that even when mastered requires a total time of many hours, it's best to break the elements into subsets, manufacture, and test several teros. Before you start an implantation experiment.
It's important to remember to pretest a tero for proper inter wire and individual impedance and to take extreme clear when implanting and secure thresholds in the brain. Methods like automated real-time tracking can be incorporated to simplify the analysis procedure. After watching this video, you should have a good understanding of how to manufacture and implant teros in order to coordinate physiological activity with the complex behaviors of a freely moving insect.
