This technique demonstrates how an organism detects its environment through mechano sensory stimuli and how the information is integrated and transformed in higher centers of the CNS by exposing the ventral nerve cord and the circle nerves. First one records from the connectives in the ventral nerve cord, which demonstrates the type of signals the CNS is conveying. Next one records from the primary sensory neurons to compare the activity of primary neurons with the activity in the connectives.
The results show how movement of the fine hairs by air vibration relates electrical signals to the CNS and how those signals are processed. This method can provide insight into the integration of sensory neuron activity and cockroach, or it can be applied to other motor circuits or other model organisms. Generally, individuals new to this method will struggle because dissect can be a bit challenging to dissect the animal just right to keep the sensory hairs dry while recording from the primary sensory nerve.
Visual demonstration of this method is critical as it's difficult to learn because it's easy to damage the nerve bundle. Select a male cockroach from the holding tank that has robust circa the last segments of the male are narrow and have no ovaries and egg mass, unlike the female. The P Americana males also have a pair of short styl eye between the circa, not observed in the females, cut off the wings, legs, and head.
Then pin the body ventral side up to a dish lined with silicone elastomer with forceps. Pick up the ventral plates and cut them off with fine scissors starting at the posterior end and working anteriorly Always keep the internal organs moist with wringers while trying to keep the circa dry. This is done by using wax or rubber to raise the abdomen.
If the Cirque do get wet, dry them with a piece of tissue paper. Next, push aside the internal organs and white matter. The VNC should now be visible between the shiny trache E.It runs the length of the abdomen.
The lighting may need to be adjusted to see the VNC. Do not handle the VNC with forceps or insect pins. Instead, manipulate it using glass probes.
Clear away the animal's trache E system from the nerve cord as best as possible with forceps with a pair of fine glass needles. Very carefully split the VNC connectives longitudinally between the A six and a five or the A five and a four ganglia cradle. The Siri and abdomen upwards out of the saline bath.
Use shortened insect pins and wax or a wedge of the silicone elastomer. Be extra careful in the last abdominal segment to avoid damaging the circle nerves, which project into the ganglion. The dissected preparation microscope and recording apparatus should be set up inside a Faraday cage to block external electric fields that could override signals from the neurons.
Position the microscope so that it is overlooking the stage. Then adjust the position of the high intensity illuminator beam. To best visualize the preparation, connect the AC CDC differential amplifier to the integrated data recording unit.
The head stage holding a micro electrode should be connected to the amplifier. To stabilize the recordings. Ground the abdomen with the silver wire coated with chloride.
The reason for the silver ground wire is if the solution inside the body cavity loses contact with the bathing fluid in the dish. Then the solution which is associated with the recording electrode remains grounded. Next, set the recording frequency to four kilohertz.
Then start the voltage scale on the trace at 500 millivolts. Adjust this as needed and set the software to run in either continuous or oscilloscope mode to the preparation. Cut one of the VNC connectives close to a five.
Then place the cut end attached to a six into a suction electrode prefilled with ringer solution. With a dry pipette blow air onto the hairs located on each circus. Check if stimulating the hairs on the circus ipsilateral to the recorded connective gives a different response than the contralateral one.
Take note of the response amplitude and the frequency of the spikes. Then move the suction electrode to a circle nerve for recording. If the fit isn't good, switch to an electrode tip with a smaller opening.
Next, cut the circle nerve close to a six and suck up the nerve leading to the circus. This should result in spontaneous action potentials. Now blow air onto the circus and note the responses.
Switch the recording software to sweep mode so that it records traces each time a stimulus is triggered. These traces should be between one and 500 milliseconds. Connect the stimulating electrode to the output of the stimulator and connect the BNC trigger output from the stimulator to the trigger input on the recording unit.
Now, certain stimulation parameters should evoke a response, adjust the stimulating voltage to obtain a signal just over the threshold and able to obtain a maximal response. There is no reason to go to the voltages much higher than the maximal threshold for recruitment. As a high voltage can be damaging to the nerve, Cut the circle nerve as distal as possible so that a long nerve root can be pulled into the stimulating suction electrode road.
Now, the connective between the A six and a five ganglia or in a more anterior segment may be used. Draw saline into the suction electrode and then pull up a cut connective into the electrode. Ensure that the stimulating electrode is grounded in the bath saline.
Ideally, the recording electrode should be in the abdomen near a three. Now deliver a series of single stimuli of increasing voltage until a compound action potential appears on the screen. Make note of the minimal stimulating voltage and duration to recruit a response.
Start low and increase the intensity until a synaptic response in the connectives is observed. The large spike and extracellular action potential from the giant axons will appear first, and then other smaller action potentials may also be observed by puffing air at the cci. Large spikes were observed in the connectives using this stimulating paradigm.
Recordings from the connectives between a three and a four typically showed a large spike characteristic of the giant inter neurons recording from a circle nerve while physically rubbing the Siri with tweezers produced a strong burst of activity in another recording two puffs of air, each produced a rapid bursting response in the circle nerve when electrically stimulating the circle nerve with a suction electrode and recording in the connective between a three and a four, a threshold in the stimulation to evoked responses was recorded. Electrical stimulation of the circle nerve clearly elicits a response in connectives that can be quantified for manipulative studies with pharmacological agents or with local environmental parameters Once mastered, this technique can be done in about an hour if it is performed properly. While attempting this procedure, it's important to remember not to pinch the nerves with the tweezers or not to pull the nervous too hard with suction electrode.
After watching this video, hopefully you have a good understanding of how to dissect the cockroach nervous system and collect sensory evoked extracellular recordings.
