The opener muscles obtained from a crayfish walking leg, which is automized from the animal. Once the muscle is exposed, the innovation pattern can be revealed by various staining procedures. Synaptic responses in the form of short-term facilitation are readily recorded from the muscle.
A focal macro patch electrode can be placed directly over identified varicosities along the nerve terminals. Synaptic responses are then recorded, which can be used for measuring synaptic efficacy at a qual level from a defined location on the terminal. Hi, my name's Ann Cooper from the lab of Dr.Robin Cooper in the Department of Biology at University of Kentucky.
Hi, I am Robin Cooper. My student and I will show you a procedure to dissect the crayfish opener muscle and use it for recording synaptic responses with different stimulating paradigms. We use these procedures in our laboratory to study synaptic efficacy and the relationship between synaptic ultra structure and function.
So let's get started. Back in 1879, the zoologist TH Huxley wrote a book entitled The Crayfish, which is still regarded today as a comprehensive guide to the life history, anatomy and physiology of this crustacean. Around this time in history, the innovation of the opener muscle in crayfish legs was being characterized, and physiological studies were already underway in muscles of the crayfish.
In fact, the experiments in crayfish might possibly be the first to demonstrate facilitation at the neuromuscular junction or NMJ. Dr.Riche published his primary research in his medical textbooks, which he used to teach medical students about muscle function. Over the next few decades, crayfish n mjs were being described anatomically and physiologically in respect to tension development and anatomy.
In 1961, doodle and Kler demonstrated facilitation in the crayfish opener muscle and showed for the first time the phenomena of presynaptic inhibition and reported on the qual nature of synaptic transmission. At this NMJ in 1971, Sherman and at Edward made the seminal discovery that the opener NMJ in crayfish exhibited long-term facilitation or LTF. In addition to short-term facilitation or SDF.
From this period on, many investigators focused on the attributes of SDF and LTF using the opener NMJ of crayfish to study the cellular mechanisms. Today, the opener muscle preparation in crayfish is still used to study the fundamentals of synaptic transmission because it is easily accessible and very hardy in comparison to many others. Synaptic preparations to obtain the crayfish leg for dissection, a crayfish six to 10 centimeters in body length is induced to automize its first or second walking leg by forcefully pinching at the Iscu pod segment.
This procedure does not kill the crayfish which has returned to its holding tank. Place the crayfish leg on a piece of tissue paper on the microscope stage. Turn the leg around until the outside is facing up.
This is usually the arched side up. Use a sharp razor blade to etch the cuticle until just cutting through. In this pattern, be careful not to cut too far distal on the dorsal to ventral cut by the marrow pod to copper pod joint.
Leave the cuticle in place for now with the razor scalpel blade etch the cuticle on the proper diet until just cutting through in this pattern for the proper diet segment on one side. After that, repeat on the other side, joining the proximal cuts to avoid cutting into the opener muscle, keep the blade leaning to the closer muscle. When cutting through the cuticle, leave the cuticle in place for now.
Next, submerge the leg into crayfish saline in a dissection dish with the one centimeter thick sill guard coating on the bottom. Stick a pin in the dorsal cordal corner within the cut of the window made in the meite to hold the preparation still with fine tweezers. Lift the cuticle slightly from the distal end, and with the razor cut the flexor muscle fibers away from the cuticle in a distal proximal manner.
Then lift the window of cuticle off. Once the cuticle is lifted off, carefully pull the tendon away from the leg cavity and cut the AEM at the meite to CARite joint. Take care to only cut the tendon and not the main leg nerve that is on the inner side of the tendon.
Pinch the tendon where it was cut with tweezers and pull the flexor muscle off by lifting it in a cordal direction. The main leg nerve and the extensor muscle are now exposed. Proceed to the proper diet segment and cut at the proper diet to dactyl lipid dite joint.
Pull the ventral segment of the proper diet down and back quarterly so that the muscle attached in the cordal region could be seen. Cut these muscles with the razor. Be careful not to cut the motor nerve branch to the opener muscle.
The opener muscle is now exposed to the saline. Return to the most cordal region of the meite segment where the leg nerve bundle usually contains two separated nerve bundles. Transect the dorsal bundle with fine scissors.
Then pick up the cut end with tweezers and gently pull distally until about half the length of the meite segment is reached. This long nerve branch contains the excitatory opener nerve. While the larger bundle of nerves contain the inhibitory motor neuron of the opener muscle.
Cut the preparation in the meite segment in a diagonal manner such that an insect pin can be placed through the dorsal aspect of the meite. This positions the ventral aspect of the opener muscle up so that it faces the observer. Next, remove the residual fibers of the closer muscle that blocks the view of the opener muscle.
By pushing the fibers against the cuticle and outta the cavity, the main leg nerve that runs along the opener muscle and goes into the dactyl lipid can be pulled up gently with the fine tweezers and then cut away. Now the opener muscles is exposed without any tissue to get in the wave. An intracellular electrode or a focal macro patch electrode to recall the excitatory post-synaptic potentials or EPSPs place the opener muscle preparation in a recording chamber designed with a plastic suction electrode.
Having the stimulating electrode built into the chamber avoids having to use a micro manipulator to place a stimulating electrode pin the preparation down in the recording dish and place the branch of the nerve that contains the excitatory nerve in the suction electrode. The opener muscle is divided into three general regions, distal, central, and proximal. Although the entire opener muscle is innovated by a single motor neuron, the njs are structurally different and have regional specific differences in synaptic efficacy.
Since the most distal fibers are easily demarcated for consistency among preparations, we will use the distal region for this demonstration to elicit an evoked response. The excitatory axon will be selectively stimulated by a grass eight eight stimulator. The distal region of the opener muscle is impaled with a sharp intracellular electrode filled with three molar potassium chloride.
A model axon clamp two B amplifier, and A one XL lu head stage is used for recording intracellular EPSPs. Short-term facilitation or various other types of responses desired, can be obtained by varying the stimulus conditions. STF is obtained by giving a train of 10 or 20 pulses at 10 or 22nd intervals respectively to the excitatory nerve.
The frequency of stimulation within the train can be varied. The output of the grass is connected to a stimulus isolation unit with the lead connected to the silver wire that is within the plastic suction electrode. The lead is attached to a silver wire in the bath to record focal qual EPSPs directly over identifiable regions of the nerve terminal.
The synaptic varicosities are first visualized with a vital fluorescent dye four dye two A SP.A macro patch recording electrode made from chix glass will be used here. The lumen of the electrode is filled with a saline bathing medium working under fluorescence microscope, placed the lumen of a macro patch recording electrode directly over a single isolated varicosity to evoke the nerve terminal. The excitatory motor nerve is stimulated as demonstrated earlier.
Spontaneous, as well as evoked qual responses can be recorded along the string of visualized varicosities by gently lowering the electrode lumen and raising it over. Each varicosity direct counting of qual events is possible with low stimulation frequencies for each evoked response. The number of qual events can be determined for a series of responses.
The total numbers of qual events are counted to then estimate mean qual content based upon these direct counts. One approach to calculate mean qual content is to take the total number in the types of evoked qual events during stimulation and divide by the total number of stimulus trials using the following formula. Shown here is a set of repetitive trials of stimulating 30 pulses at 40 hertz.
Note the facilitation for each run and the average response if the stimulation has increased to 60 hertz. More pronounced facilitation is observed when recording with a focal electrode over a varicosity and stimulating at one hertz. The evoked qal responses are observed.
We just showed you how to dissect the crayfish opener muscle and use it for recording synaptic responses with various stimulation paradigms and techniques. When doing these procedures, it's important to remember to treat the neurons with care and not to pull on them too hard or to pinch them. Also, when you're placing the focal macro patch electrode on the visualized varicosities to lightly place the electrode on those varicosities and lift it back off.
One other point to remember is that you don't want to expose the preparation too long to the mercury light as that could damage the nerve terminal as well. So that's it. Thank you for watching and good luck with your experiments.
