TENSION RECEPTION OF A SKELETAL MUSCLE IN A CRAB LEG
By
Zana R. Majeed1,2, Josh Titlow1, H. Bernard Hartman3, Ellen Burns1 and Robin L. Cooper1
1Department of Biology, University of Kentucky, Lexington, KY 40506, USA; 
2Department of Biology, College of Sci, Univ. of Salahaddin, Erbil, Iraq;
3Oregon Institute of Marine Biology, University of Oregon, Charleston, OR 97420, USA

1. Purpose

To understand how primary sensory neurons convey force information that is generated by a skeletal muscle of an animal. In addition, one will learn some gross and fine anatomy by dissecting and viewing alive neurons under the microscope. The electrical activity from a joint receptor organ will be recorded by using basic neurophysiological equipments.

2. Preparation

Opener muscle and leg from the blue crab Callinectes sapidus.

3. Introduction

Monitoring the force skeletal muscles exert is an intricate and complicated task as there are unique morphological arrangements, such as, various pinnate and fusiform structures of muscle fibers. Also, muscles in different developmental stages generate various force and some are capable of producing a wide range of forces after maturation. Vertebrates use Golgi tendon organs (GTO) that are strategically placed in attachment points of tendons and muscles to monitor factions as well as whole muscle force (Hill, 1970; Stuart et al., 1972). Generally, it is assumed that these sensory structures act to protect muscles and tendons from overuse and damage (Hill, 1970; Jami, 1992; Mileusnic and Loeb 2006). However, it is a daunting task to monitor each GTO in a mammalian muscle and related the forces exerted on each sensory neuron in relation to regional and total muscle tension. Perhaps less sophisticated skeletal muscular systems offer an advantage for such discrete measurements. In fact, studies in which one can ablate different types of skeletal muscle fibers in a mixed muscle (glycolytic or oxidative) or by reducing various regions through removing tissue would be an asset to future understanding of GTO physiology and functional significance in their arrangements within muscle-tendon units. 
As in the vertebrates, invertebrates also have a need to measure the force that a muscle can generate to avoid muscle or cuticular structural damage. In arthropods, receptors that mimicked GTOs were first described by Macmillian and Dando (1972). This early study in invertebrates was performed in a leg of a crab (Cancer magister) and subsequent studies have reported skeletal muscle tension receptors analogous to GTOs in several invertebrates including insects (Carcinus meanas- Parsons, 1980, 1982; Callinectes sapidus-Hartman, 1985; Tryba and Hartman 1997; Limulus-Eagles and Hartman, 1975; Eagles and Gregg, 1984; Locust legs-Theophilides and Burns, 1979).
Arthropods do not have an internal skeleton for skeletal muscle attachment. Instead they have developed an interesting means of making internal attachment points from invaginated exoskeleton that can form a tendon like structure for skeletal muscles to attach. These structures are referred to as apodemes. The sensory ending of the tension neurons are embedded within the layers of the apodeme that appear to monitor the shearing between the layers within the cuticle of the apodeme (Parsons, 1980). Skeletal muscle fibers are attached to the apodeme and then span a region and attach to the internal side of the exoskeleton. Thus, when the skeletal muscle contracts, it will pull apodeme in relation to the exoskeleton. The apodeme association with the exoskeleton occurs across a joint in which the skeletal muscle resides. Thus, joints are moved by the pulling on the apodemes via the skeletal muscle. In the apodemes examined to date within the walking leg of the crab C. magister, it appears that each has a set of associated tension receptors (Cooper and Hartman,1994). At least in the crab leg, the tension receptors always appear to be most distally associated within the apodeme. In addition, the tension sensory nerve bundle is observed generally close to where the chordotonal nerve joins the main leg nerve bundle (Cooper and Hartman,1994).    
In this exercise, one should think about what physiological criteria are used to describe tension receptor and how to obtain the measures to prove if the neurons you will record from are monitoring the forces from skeletal muscles (Hartman, 1985).

4. Methods
4.1 Materials
	1. Faraday Cage
	2. Micromanipulator
	3. Suction Electrode
	4. Dissecting Microscope
	5. High Intensity Illuminator (light source)
	6. Microscope Platform
	7. AC/DC Differential Amplifier (A-M Systems Inc. Model 3000)
	8. PowerLab 26T (AD Instruments)
	9. Head stage
	10. LabChart 7 (ADI Instruments, Colorado Springs, CO, USA)
	11. Crab saline (see Table3)
	12. Methylene blue: This is made in crab saline at a concentration of 0.25%
	13. Sylgard coated dishes (Dow Corning, SYLGARD® 184 silicone 			 		elastomer kit; Dow Corning Corporation, Midland, MI. USA)
	14. Dissecting tools
	15. Big and small insect pins
	16. Glass rod/tools for dissecting and manipulating nerves
	17. Pipettes and beakers
	18. Force transducer

4.2 Setup

Figure 2: The equipment set up

1. Setup up the Faraday cage. The microscope, high intensity illuminator, micromanipulator, and the saline bath will all be set up inside the cage (The Faraday cage is used to block external electric fields that could interfere with the electrical recording).

2. Setup the microscope in a position where it is overlooking the microscope stage.

3. Position the high intensity illuminator in a convenient position.

4. Prepare a saline bath using crab saline in a Sylgard dish and place it under the microscope (this is where the dissected leg will be placed).

5. Position the micromanipulator in a position where the suction electrode has easy access to the saline bath.

6. Suction up saline until it is in contact with the chloride coated silver wire inside the suction electrode. Arrange the other wire on the cut-side of suction electrode close to the tip of electrode, so both wires will be in contact with the saline bath. 

7. Connect the AC/ DC Differential Amplifier (amplifier) to the Power Lab 26T. Do this by connecting the proper cord from Input 1 on the PowerLab 26T to the output on the amplifier.

Figure 3: Extracellular amplifier

The settings for the amplifier are as follows:

CONTROL	SETTING	
High Pass	DC	
Notch Filter	OFF	
Low Pass	20kHz	
Capacity Comp.	Counterclockwise	
DC Offset Fine and Course knob	Counterclockwise	
DC Offset (+OFF)	OFF	
Gain knob	50 (to start with)	
Input (DIFF MONO GND)	DIF	
MODE(STIM-GATE-REC)	GATE	
ΩTEST	OFF	

8. Connect the head stage to the ‘input- probe’ on the amplifier.

9. Connect the electrical wires from the suction electrode to the head stage. The wires should be connected with the red (positive) at the top left, green (ground) in the middle, black (negative at the bottom. This is indicated in Figure 4. The ground wire can just be put in the saline bath. 

Figure 4: Head stage Configuration

10. Now connect the USB cord from the PowerLab 26T to the laptop. Ensure that both the amplifier and PowerLab26T are plugged in and turned on before opening LabChart7 on the computer. 

11. Open LabChart7.

	-The LabChart Welcome Center box will open. Close it.
 	- Click on Setup
-  Click on channel settings. Change the number of channels to 1 (bottom left of box) push OK.
-At the top right of the chart set the cycles per second to about 4 KHz. Set the volts (y-axis) to about 500 or 200mv.
-Click on Channel 1 on the right of the chart. Click on Input Amplifier. Ensure that the settings: differential, ac coupled, and invert (inverts the signal if needed), anti-alias, and differential are checked.
	- To begin recording press start.

12. Force transducer 

Recording forces of contraction in excised preparations 

Figure 4: Setup

12.1. Attach the transducer to the bridge pod.

12.2 Attach the bridge pod to the PowerLab 26T.

12.3. Attach the PowerLab 26T to the USB port on the computer.

12.4. Open LabChart7, by clicking on the labchart7 icon on the desktop.
- The LabChart Welcome Center box will pop open. Close it.
- Click on Setup
-  Click on channel settings. Change the number of channels to 1 (bottom left 		of box) push OK.
-At the top left of the chart set the cycles per second to about 4KHz. Set the 		volts (y-axis) to about 500 or 200 mV.
-Click on Channel 1 (or what ever port the transducer is plugged into)  on the 		right of the chart. Click on Input Amplifier. Ensure that the settings: 			single-ended, ac coupled, and invert (inverts the signal if needed), and 		anti-alias, are checked.
- To begin recording press start.

12.5. Gently move the force transducer pin to see that the movements are detected in 	the recording 

12.6.  Place the force transducer in the clamp near the dissected crab leg
    
12.7.  Hook the force transducer in the as shown in Figure ?.

Figure here of leg and transducer hooked on.

4.3 Dissection
The procedures described by Tryba and  Hartman (1997)  are being followed for the anatomical dissection and electrical stimulation. The equipment for the electrophysiological recordings is different so we have provided a detailed description of this laboratory manual.
In brief, Tryba and  Hartman (1997) obtained a walking leg of the swimming crab Callinectes sapidus caught in the Gulf of Mexico. Using a net obtain a crab from a holding tank and place into an empty bucket. While holding the crab with the net or an object across the carapace from behind, and avoiding the claws, cut across the merus of a walking leg with a stout pair of scissors. The animal will autotomize the remaining basal portion of that limb. If the crab does not break the leg off, help it by inserting the scissor tips into the wound and twisting the stump but be careful not to damage the leg. Return the animal to its aquarium.
Put the leg in the Sylgard-lined dissecting dish and cover it with the species correct cooled (12-19oC) crab saline (Table 1).

Table 1: Solution for C. sapidus saline (Blundon, 1989)

SALT				 g/l		 g/3l			 g/2l
NaCl				27.47		82.41			54.94
KCl				 0.59		 1.77			 1.18
MgCl2 6H20			 1.421		 4.269			 2.842
CaCl2 2H20			 2.205		 6.615			 4.41
Dextrose			 1.982		 5.945			 3.964
HEPES acid (5mM)		 1.19		 3.57			 2.38
HEPES salt (5mM)		 1.30		 3.90			 2.60
Adjust to pH 7.5 with NaOH (2N) or HCl (1M). The osmotic pressure should be 1000 mm/l.

The lateral and medial side of the leg should be determined. The medial side has a soft texture that can be felt by pinching gently in the merus region with a fingernail. Face this soft cuticle side up in the dish. The excitor motor nerve that innervates the opener muscle also innervates the stretcher muscle in the carpus. In order to stimulate the opener muscle, the stretcher motor nerve should be isolated in the carpus region and then stimulated. With a pair of scissors, make an incision through the merus. Discard the leg proximal to the cut. Pin the preparation in the proximal part of the carpus and in the dactylus. Remove a section of the cuticle in the carpus region and find the nerve branching from the main nerve bundle to the stretcher muscle. Tease a section of the nerve that projects towards the opener without pinching the nerve. The opener motor nerve is transected to pull it into the suction electrode and then stimulated. Make sure that the saline is up to the wire that is located inside the suction electrode; otherwise, electrical activity cannot be obtained.
Use Figure 5 to guide you through the rest of this dissection. Cut off the closer muscle with a scalpel with a #11 blade in the propus. (Note: Do not cut deeply in the proximal region because it might damage the opener motor nerve). 

Figure 5: Cut along the dotted line on the propodite

The saline should be exchanged with fresh cooled saline throughout the dissection process to keep the neurons alive. For further dissection, place the preparation dish under a dissecting microscope and use fiber-optic illumination.
, In the next step, carefully cut the closer tendon from its attachment to the dactyl using sharp-pointed medium-size scissors. Be very careful not to disturb the main leg nerve which should be clearly visible, remove and discard the opener muscle and tendon. This will require minimal cutting and trimming. Make a hole in the dactylus with a dissection pin. This hole will be used later, but it is necessary to make it while the leg is in this position.
Locate the nerve branch that projects to the opener muscle and apodeme in the distal region of the opener muscle. Carefully probe with the glass tool. Ask for help if you cannot find the nerve bundle. Now pin the main nerve so that the ventral view of the opener muscle is facing upward toward the observer (see Figure ?). Be cautious not to damage the opner muscle fibers. 

Figure 6: Exposed tension nerve .???

 4.4 Recording
One is now ready to determine if the preparation is still alive. Electrically ground the bath. Change the saline often with saline that has been kept on ice. Turn on Lab chart recording software. To detect neural activity, draw the cut end of the opener tension nerve into the recording suction electrode.

The first set of experiments is to examine for passive tension on the opener apodeme.
 
(i) Is there a patterned and consistent response to the opening and closing movements of the dactyl with passive movement? 

Next we will test for active force development related to stimulation frequency.

(ii) Firmly attach a metal hook so that the tip of the hook goes through a small hole in the dactylus. The other end should be attached to the force transducer. The metal rod should be strong enough that it does not bend easily. Pull the hook and dactylus so that it is about a 45 degree angle of the opener joint. Now stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve. Repeat this process twice. Next extend the joint 180 degrees. There maybe some force measured by the transducer due to this passive stretch of the muscle. Now stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve. Repeat this two times. Next bend/flex the joint so that it is fully flexed (~90 degrees). Now stimulate the motor nerve at 100 Hz for 250 msec and measure the force as well as the firing frequency of the tension nerve. Repeat this two times. NOTE: Make sure the transducer is at a 90 degree angle each time so that the pin is perpendicular to the transducer for maximal detection of the force generated (see figures ?, 45, 180, 0 degree placements).

(ii) What types of responses are evoked by pinning the dactyl at various fixed positions? 
(iii) Is that response consistent when repeated? 

By filling in Table 2 with values the observations will be easier to make into quantitative statements.

Table 2: Static position. Firing frequency measures
Angle(	Time in recording before activity appeared after stimulation started	# of Action Potentials in 250 msec stimulation period	# of different sized action potentials in  250 msec stimulation period	
180 
(extended)				
				
45				
				
90
 (flexed)				

(iv) Now measure the response with a quick tension release. Arrange the preparation so that the hook on the tension transduce can easily be pushed out of the hole in the dactylus. Bend/flex the joint so that it is fully flexed (~90 degrees). Now stimulate the motor nerve at 100 Hz with a continuous stimulation of 5 seconds. After the first second or less, as tension has built up on the opener muscle, push the pin out of the hole in the dactylus.  Examine the tension nerve recording before and after release of the pin holding the dactylus. Write down your observations.

Make careful notes of the kinds of responses observed. After you are satisfied with your observations save the chart file and take a photo with your phones of the preparation. 
You may wish to determine if the neuromodulators octopamine, serotonin, and proctolin alter the output of the tension neurons by adding these compounds to the bathing media over the exposed opener muscle and repeating the experiments. Use a pipette and just drip over the preparation (range 10µM to 0.5mM).

4.5 Staining
Cobalt back-fill of the tension nerve:
Using the in situ preparation use the long tension nerve according to the following procedure. A one-day back-fill in the incubator at 13 (oC) is suggested.
Expose the nerve. Keep it immersed in cold saline that is refreshed every 10-15 minutes during the operation and in all the steps to follow except where noted. A Vaseline well is required to be made to hold the CoCl2. If CoCl2 spills into the saline bath, the entire preparation will stain black. One needs to slip a small cut of a polystyrene sheet and pin it in such a manner that it will not float away or become immersed in the saline bath (Figure ?).

Figure here

Ejecting petroleum jelly (Vaseline) or silicone stopcock grease from a fine hypodermic needle fastened to a disposable syringe, make a barrier (a circle might work well) on top of the slip of polystyrene. The barrier should be about 1-1.5 mm high except for a shallow "V" at midpoint where the nerve will be draped across. Make a small puddle of saline on either side of the barrier near the "V". Taking care not to stretch or pinch the nerve. Lift the nerve carefully from the dish and place it in the saline puddle in the Vaseline well. This procedure should be carried out quickly so that the section of nerve does not dry out. Carefully eject Vaseline or stopcock grease to cover the exposed nerve. Now test the barrier with saline and make sure it is not leaking. To assure yourself that the two sides are isolated from one another, blot away saline on the inside of the barrier and see if it fills up when the bath saline is high around the wall of Vaseline. If saline does not leak across in a few seconds, the barrier is probably sealed (Figure ?).

Figure here
 
Using the rolled up point of a piece of tissue paper, blot up the saline in the Vaseline chamber without the paper wrapping up the nerve. Make a new puddle using a few small drops of distilled water, and then cut the nerve end. Be very careful not to pull the nerve cord through the barrier when making the cut. The osmotic shock of the distilled water will "balloon" the axons of the connectives. Within 30 seconds add a small drop of CoCl2 to the water, blot up this solution, and then add enough CoCl2 to form a puddle over this shortened section of nerve. Preparations are best kept refrigerated at 4oC for 12-24 hrs (Figure ?).

Figure here

The rest of the procedure involves precipitation of CoCl2 which has been taken up by the axons and transported to neural processes and cell bodies. Remove the humidifying puddles. Blot away the cobalt solution using a tissue and wash away the remnants of cobalt with several changes of saline. Transfer the isolated nerve cord to a small glass Petri dish containing about 10 ml of saline. The neurons are washed and the following steps are done in situ. Good metal tools are not to be used to handle the preparation after this step. Add 1-2 drops of ammonium sulfide (NH4)2S to the saline. Cap the bottle tightly and place it back into the hood. Observe the reaction in the preparation under a dissecting microscope (Figure ?).
Within a minute or two, cobalt filled neurons and their processes should begin to appear. After 5-10 min, replace the development solution with fresh saline but make certain that the development solution you have poured into the sink drain is followed by tap water for a few minutes.

Figure here

Pour out the saline and fix the nerve preparation for about 15 minutes with two changes of Bouin’s solution fixative (Sigma). For larger tissues, increase the duration of fixation. Dehydrate in an ascending order of ethanol concentration beginning at 70% (i.e. 70%, 80%, 90%, 100%). About 10 minutes at each concentration is sufficient for small tissues. After about 10-15 minutes in two changes in 100 % alcohol clear the tissue by replacing alcohol with methyl salicylate. The preparation will stay in this solution permanently for repeated viewing. With time the filled cells will become more apparent because the surrounding tissue will become clearer.
5. Results

	Representative anatomical morphology is illustrated in the Methods in hopes of helping to provide an understanding of the anatomy so that physiological recordings can be made without the use of stains or dyes. Sometimes, one can observe the tension nerve better when moving the light at various angles and intensity to obtain the best contrast. Tracking the tension nerve close to the muscle fibers, at the distal of the opener apodeme, maybe help one to observe the branch from the main leg nerve. Figure ? illustrates the tension nerve separated from the main leg nerve. 

Tension nerve activity:
-fully extended,
-45 degress
-Fully flexed
---full extended and various stimulus frequencies..20Hz, 40Hz 60 Hz..short bursts for 5 seconds

Force recordings:
-fully extended,
-45 degress
-Fully flexed
---full extended and various stimulus frequencies..20Hz, 40Hz 60 Hz..short bursts for 5 seconds

Together if possible or superimpose the recordings (depends on electrical noise from the transducer)

6. Discussion

REFERENCES:

Tension references need to go here

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