The overall goal of this procedure is to dissect the abdominal nerve cord from the crayfish in order to use it in electrophysiological studies. This is accomplished by first anesthetizing the crayfish. Next, a gross dissection is carried out to isolate the ventral plate of the crayfish.
Then a fine dissection is performed to completely isolate the central nervous system from the raic ganglia to abdominal ganglia. A six. Finally, the ganglion sheath is removed from all ganglia to facilitate electrophysiological experiments.
Ultimately, the chain of abdominal ganglia are used for extra or intracellular recordings. To study coordination of central pattern generators or to investigate the morphology and the synaptic context of identified neurons. This method can help answer key questions in the neuroscience field, such as providing hands-on experience on nervous network function to be able to record fic FLA commotion in the isolated nervous system, or to characterize the morphology of abdominal ganglia or that of specific neurons.
Though this method can provide insight into the swim rate system of crayfish. It can also be applied to other systems such as a neuronal network that drives the tail flip behavior in crayfish Working with the crayfish Pacific Stacia Linea Guus. After anesthetizing an animal according to the text protocol, holded ventral side up and use strong scissors to cut both claws at their bases near the thorax.
Then remove the left and right euro pods. Place the animal ventral side up in a dissection dish lined with black sard before using cold saline to perfuse through the claw opening. Next, use strong scissors to make a single transverse cut just posterior to the cray fish's eyes to decapitate it.
Then remove all the walking legs near the base joints to isolate the abdomen along with the last thoracic segments from the rest of the thorax. Insert the tip of the scissors into the opening of the second walking leg and cut to the opposite side. After extending the cut to both sides through the thorax and exposing some of the internal organs as described in the text protocol, remove the anterior part of the thorax.
Now remove the internal organs and rinse the animal with cold saline. Continue the dissection with a cut at a maximal lateral position between the lumon and the swimmer rats through the whole length of the sternal plate. Make the same cut on the other side.
Place the cray fish's abdomen ventral side up in the dissection dish. Use insect pins to fix the specimen posteriorly at the tellson and anteriorly at the remains of the carabase. Fill the dish with crayfish saline so that it covers the specimen.
Use coarse forceps in the left hand to grab through a walking leg opening and pull the specimen open. Identify the large two dorsal flexor muscle strands. Fix them and cut their ventral bases as well as the sternal artery.
Expose the first abdominal ganglia A one and associated nerves N one N two. Now moving to the second abdominal segment. Remove the basis of the muscle strands close to the ventral basis.
The abdominal ganglion A two is now visible with the associated nerves N one, N two and N three. Transect the nerves N three at the most distant position from the nerve cord. Repeat these steps for the next three abdominal segments with the abdominal ganglia.
A three to a five. When reaching the abdominal ganglion, a six. Cut the sternal plate posterior to the nerves of a six.
Keep the ventral part and pin it down. Dorsal side up in the dish filled with saline under the microscope. Use forceps to remove the most anterior parts of the cephalon thoracic stern.
Then cut the muscles between the remaining exoskeletal structures. After carefully lifting the anterior end of the ventral nerve cord, cut the thoracic nerves laterally. Focus on a one and cut the nerves N one and N two of a one at a maximum of one centimeter in length for pinning them out.
Next, move to A two and identify the N one, N two and N three nerves of this segment. The nerves n one of abdominal ganglia, a two to a five. Reside between two sternal cuticular in unfoldings in each segment and are covered by musculature.
Then beginning at the lateral rim of the abdomen, make one cut along the posterior sternal cuticular infolding, and proceed towards the midline After uncovering N one according to the text protocol. If necessary, cut the nerve as distally as possible. Proceed to the contralateral side and begin isolating the nerve N one from the midline near the ganglion proceeding towards the lateral rim by cutting along the posterior sternal cuticular enfolding.
Now cut the nerves N two of this ganglion to a suitable length for pinning. Then repeat the steps shown for abdominal ganglia. A two for a three to a five as distally as possible.
Cut the nerves of ganglion a six. Use forceps to grab multiple nerves of a six to lift the ganglion and start isolating the chain of ganglia from the sternal plate. After completely isolating the nerve cord, transfer the chain of ganglia to a petri dish lined with clear sill guard and filled with saline using small pins cut from stainless steel wire while applying a gentle stretch touch.
Pin the chain of ganglia in a straight line with the nerve cord arranged dorsal side up. Keep only the last two thoracic ganglia and pin the thoracic nerves to the side. Pin out the nerves of a one at a 90 degree angle relative to the nerve cord.
Then proceed to a two and pin the nerves N two at a 35 to 45 to degree angle relative to the nerve cord. Once the nerves N one are separated and pinned according to the text protocol, pin the anterior branch of the nerve N one at a 90 degree angle to the nerve cord. Then pin the posterior branch of nerve N one between the anterior N one branch and the nerve N two.
Since all abdominal ganglia have to be des sheathed, use fine spring scissors to make a small lateral cut through the ganglion sheath posterior to the ganglion and between the nerves N two and N three. Cut transversely across the sheath above the connective. Then continue to cut it along the lateral borders of the ganglion before removing the sheath.
If necessary, use fine forceps to lift the sheath to carry out extracellular recordings. Following the setup of tools and reagents listed in the text protocol, place the chain of ganglia on the microscope table and illuminate from below. Insert the recording electrode into the syl guard close to the target nerve and the reference electrode at a position nearby but lateral to the ganglia.
Then bend the target nerve around the recording. After stretching and securing the nerve, use a syringe filled with petroleum jelly and a rounded tip 20 gauge needle to isolate the target nerve from the bathing solution. Repeat this for all nerves whose activity needs to be recorded.
Finally, begin recording as shown here, the extracellular PS activity of ipsilateral PS two to PS five motor neurons starts with a PS burst in a five and the excitation wave propagates in the anterior direction. In addition, the time measured from the onset of one burst to the onset of the next burst in each segment remains constant and is defined as the period of the rhythm periods and burst. Durations can vary between different preparations, but the phase lag between segments remains constant at approximately a quarter of the cycle independent of the frequency of the rhythm.
These coordination patterns can best be seen in the phase histograms seen here. Here using sharp micro electrodes, intracellular recordings from the dendrites of Ps.Motor neurons show enphase oscillations of individual neurons with the extracellularly recorded activity of all PS motor neurons of the same hemi ganglion. To identify intracellularly recorded neurons, cells can be di filled using sharp micro electrodes and iontophoresis.
In this figure, the ventral soda of two di filled PS motor neurons are located posterior to the base of the nerve N one. The primary neurite projects anteriorly and branches within the lateral neuro pill. The axon projects through the posterior branch of the nerve N one to the musculature.
Once mastered, this technique can be done in 45 minutes to an hour if it is performed properly. While attempting this procedure, it's important to remember to change the saline at regular intervals to keep the nervous tissue healthy. Following this procedure, other methods like intracellular recording of motor neurons or inter neurons can be performed in order to answer additional questions like analyzing the cellular mechanism of coordination, measure ionic currents or study the effect of neuromodulators on rhythmic activity of specific cells.
