The overall goal of this procedure is to demonstrate the dissection of and recording from primary sensory neurons, conveying information of joint movements and positions as proprioceptive information for an animal. This is accomplished by first dissecting the abdomen of the crayfish and exposing the dorsal half of the preparation. The second step of the procedure is to record the electrical activity from a joint receptor organ by placing a segmental nerve within a suction electrode.
The third step of the procedure is to move the joint to different positions and correlate movement with activity profile. The final step of the procedure is to examine the anatomical structures associated with the sensory organ. This procedure shows an approach for recording activity from primary proprioceptive neurons and can be used to study effects of biogenic neuromodulation.
This method can help instructors teach fundamental concepts in sensory physiology, such as sensory adaptation and how neuromodulators alter sensory perception. Though This method can provide insight into proprioception and invertebrates, it can also apply to other model organisms such as mammals to better understand intrafusal muscle spindles. This procedure covers how to isolate, stimulate, and record from the muscle receptor organs located in the crayfish abdominal muscles.
The first step is to dissect the crayfish nerve and abdominal muscles containing the muscle receptor organ. Select a crayfish we are using Pro pros clarkia measuring six to 10 centimeters in body length and place it on ice for two to three minutes to anesthetize it. The dissection will begin by decapitating the crayfish as shown by lines marked A in the figure.
The next cut at B will be to separate the thorax from the abdomen, and the third cut at C will open the abdomen. Once the crayfish is anesthetized, hold the anesthetized crayfish from behind the claws with one hand. Quickly cut from the eye socket to the middle of the head on both sides, and then behead the crayfish.
The blood from the preparation is sticky when it dries, so clean the tools immediately. Once the crayfish is beheaded, cut between the thorax and abdomen on the ventral side. Cut off the swim marts.
If the crayfish is male, cut off the stylus. It should now be easy to separate the abdomen from the thorax. Place one blade of the scissors inside the abdomen, and with the scissor tips pointing away from the preparation cut along the lateral border.
Repeat on the opposite side. Take the back end of the tweezers and push the muscles and GI tract away from the dorsal side of the preparation. Be sure not to push down on the muscles.
Cut across from lateral to lateral the last rib to remove the ventral side of the tail. Submerge the preparation in crayfish, saline, and place the cigar dish containing the crayfish abdomen under the dissection scope. The next step is to set up your stimulation and recording apparatus.
The setup consists of a faraday cage, a microscope, a high intensity illuminator, a micro manipulator, and the saline bath. The Faraday cage is used to block external electric fields that could interfere with the electrical recording. Position the microscope so that it overlooks the microscope stage.
Move the high intensity illuminator to a convenient position. Insert the suction electrode into the micro manipulator and position the micro manipulator so that the suction electrode has easy access to the saline bath. Suction up saline into the suction electrode until the saline is in contact with the silver wire.
Arrange the other wire so it is close to the tip of the suction electrode, so both wires will be in contact with the saline bath. Connect the AC CDC differential amplifier to the power lab 26 T and verify the settings on the amplifier. Connect the head stage to the input probe on the amplifier.
Connect the electrical wires from the suction electrode to the head stage. The wires should be connected with the red at the top left, green in the middle, and black at the bottom. Now connect the USB cord from the power lab 2016 to the computer.
Ensure that both the amplifier and power lab 26 T are plugged in and turned on before opening lab chart seven on the computer start lab chart seven. The computer data logging software. The lab chart welcome center box pops open.
Close it. Click on setup. Click on channel settings at the bottom left of the box.
Change the number of channels to one click okay at the top left of the chart. Set the cycles per second to about 2K. Set the bolts on the Y-axis to 500 or 200 millivolts on the right of the chart.
Click on channel one. Click on input amplifier. Select the following settings, single-ended AC coupled invert to invert the signal if needed, and anti alias.
To begin data logging, click start to end data logging. Click stop electrically ground the bath by placing a silver core chloride ground wire in the bath and the other end to a common ground. If grounding, the bath causes electrical noise During recording unground the bath.
The next step is to expose the muscles and nerves of interest. The deep extensor medial muscles or DEM are identified by their twisted helix fibers and the deep extensor lateral muscles or DEL have linear fibers. This figure shows a photograph of dained DEM and DEL muscles aligned with a line drawing of the muscles.
Look at the preparation under the microscope and locate the DEM and DEL muscles. At this point, you cannot see the muscle receptor organs themselves because they're located underneath the visible muscles. Place two pins in the mid sagittal region at the distal part of the abdomen between the DEM muscles on the first and second ribs.
Look through the microscope to find the nerve carrying the sensory axons from the muscle receptor organs. Look for the segment with the most accessible nerve. The nerve is white and can be seen by using a pipette to spray saline on the preparation or by lightly blowing on the preparation.
This causes the nerve to move and thus makes it easier to identify. Once you've located the nerve, use the microm manipulator to place the suction electrode directly over the nerve. Gently pull on the syringe to draw the nerve into the electrode.
You can see the nerve being sucked into the electrode through the microscope. To begin the experiment, click start on the data logging software. Use tweezers to gently move the tail of the crayfish up and down at a 180 degree angle.
Use the comment marker in the software to note the angle of stimulation for use during later data analysis. Repeat the experiment at a 45 degree angle, recording the change of angle with the software's comment marker. Repeat at a 90 degree angle.
Notice the difference in the sensory nerve response at each angle. When you are finished recording, you can view the muscle receptor organs directly. This figure shows the relative locations of the muscle receptor organs labeled SA and RA for rapidly adapting and slowly adapting and the D-E-M-D-E-L and SEM muscles.
The photograph on the right was stained with methylene blue. To observe the muscle receptor organs in your preparation first stain the preparation with methylene blue. Pour the crayfish saline out of the cigar dish and replace it with methylene blue solution and gently swirl the dish for a few minutes.
Then pour the excess methylene blue into the waste container and pour fresh saline onto the preparation. Replace the dish under the microscope to dissect the overlying muscle. To expose the muscle receptor organs.
Cut the segment along the rib lateral to mid sagittal by placing one part of the scissors under the muscle and pulling up as you cut along the muscle. Once the DEL one and two muscles are cut, peel those muscles back and observe a thin layer of muscle called the superficial extensor medial muscle or SEM. The muscle receptor organs are the last two medial fibers lying parallel to the helix muscle.
Be very careful with this step so as not to damage the muscle receptor organs. Two distinct types of stretch receptors exist in the crayfish mechano receptor system. The phasic sensory units are innervated by fast motor axons, and the tonic sensory units are innervated by slow motor axons as shown in this figure.
When a tonic receptor is stimulated by stretch, it slowly adapts to the stimulus and continues a steady firing pattern of action potentials. In contrast, when aphasic receptor is stimulated by stretch, it rapidly adapts to the stimulus and fires only a short pattern of action potentials. This recording shows the nerve response when the abdominal segment containing it was bent.
First, the segment was bent at a 45 degree angle and relaxed. The next recording shows the response to a 90 degree flexion relaxation. This demonstrates that the rapid muscle receptor organ shows enhanced activity to a greater stretch of the MRO strand.
This next recording shows the muscle receptor organ activity invoked by flexing the tail and holding it in a 45 degree position. Note the accommodation of the neural activity shown by the decreased firing frequency. A good extension of this experiment would be to examine if neuromodulators altered the firing frequency, record the process of accommodation of the sensory response by measuring the firing frequency at various times while maintaining a given static position and entering the data in a table such as the one shown here Following this procedure.
Other methods like examining the effects of neurohormones such as topamine, serotonin, and prolactin can be tested to answer additional questions such as the cellular mechanisms of compounds on primary sensory neurons. After the development of this preparation, researchers describe the site of spike initiation in neurons and paved the way for researchers in the field of neurophysiology to explore mechanical century transduction, as well as proprioception of limbs, not only in invertebrates, but also invertebrates. This is a wonderful preparation for teaching many fundamentals in the neurophysiology.
